R Markdown

R-script for microbiome analysis following this tutorial: https://murraycadzow.github.io/2021-obss-day4/06-importing-into-R/index.html

# load the packages
library('phyloseq')
library('tibble')
library('ggplot2')
library('ape')
library('vegan')
## Loading required package: permute
## Loading required package: lattice
## This is vegan 2.5-7
# set the working directory
setwd('/Users/mandy/Desktop/16s-data-analysis/16s_R_pipeline' )

Importing raw data

# Import frequency table
import_table <- read.table('raw_data/otu_frequency_table.tsv',header=TRUE,sep='\t',row.names=1, comment.char = "")

head(import_table)
##              A1   A10   A11   A12   A13   A15    A16 A17 A18 A19    A2 A20 A21
## 0tu.1    113349 78465 68476 18916 58723 42467 177164  16  16   1 57565 447 690
## 0tu.10      706   146   166     2  5135     4   7862  42  56   9    33  49  23
## 0tu.100     175     2     0     0     0     0    989   1   2   1     1   0   4
## 0tu.1002     23     0     0     0     0     0      0   0   0   0     1   0   0
## 0tu.1007      0     0     0     0     0     0     24   0   0   0     1   0   0
## 0tu.101       0     0     0     0     0     0      0   0   0   1     0   2   2
##          A22 A23 A24 A25  A26  A27   A28 A29    A3   A30   A31   A32   A33
## 0tu.1    153 247 102 247  782  224  4851  99 71375 58889 44823 49375 25282
## 0tu.10    54   9  26  13 1916 4303 12191  20   185  8566   178  6508   290
## 0tu.100    2   1   1   1  348  155   593   0     6     8    11   163    26
## 0tu.1002   0   0   0   0    0    0     0   0     0     0     1     0     0
## 0tu.1007   0   0   0   0    0    1     6   0     0     0     0     7     0
## 0tu.101    0   0   1   4    0   32     0   0     1     0     0     0     1
##            A34    A35    A4   A5    A6    A7    A8   A9 C1 C10 C11 C12 C13 C16
## 0tu.1     7776 184008 72355  618 56973 32874 34090 1433  3   3   1   0   5   6
## 0tu.10   15260    732    22 4119    48   376  7578 4754 22 184  67 439 634   0
## 0tu.100    717     20     0 1126     0     8   814  434  0   0   0   0   0   0
## 0tu.1002     0      0    13    0     0     0     0    0  0   0   0   0   0   0
## 0tu.1007     3      2     0   13     0     0     1    2  0   0   0   0   0   0
## 0tu.101      0      0     5    0     0     0     0   25  0   0   0   0   1   0
##          C17 C18 C19 C2 C20 C21 C22 C23 C24 C25 C26  C27  C28 C29  C3 C30 C31
## 0tu.1      9   7   2  0   4   2   0   0   0   9  10    4    7   1   4   7   1
## 0tu.10   197 776  35 97  59   8 106 170 111  58 117 1295 1556 260 139 448 252
## 0tu.100    0   0   0  1   0   0   0   0   0   0   5    0    1   0   0   0   0
## 0tu.1002   0   0   0  0   0   1   0   0   0   0   0    0    0   0   0   0   0
## 0tu.1007   0   0   0  0   0   0   0   0   0   0   0    0    0   0   0   0   0
## 0tu.101    0   0   0  0   0   0   0   1   1   1   0  262  431 275   0 245 725
##          C32 C33 C34  C35 C36  C37 C38 C39  C4 C40 C5 C6  C7 C8  C9  L1 L10
## 0tu.1      1  62   0    1   2    5   3   2   5   0  7  2   0  8   3   1   4
## 0tu.10   942  77 290  948 465 1498 401 316 233 516  7  8 105 68 223 432  26
## 0tu.100    0   0   0    0   0    0   0   0   0   0  0  0   0  0   0   0   0
## 0tu.1002   0   0   0    0   0    0   0   0   0   0  0  0   0  0   0   0   0
## 0tu.1007   0   0   0    0   0    0   0   0   0   0  0  0   0  0   0   0   0
## 0tu.101   54 264 385 1153 196  464 861 590   0 468  0  1   0  0   0   0   0
##           L11 L12 L13 L14 L15 L16  L17  L18   L19   L2  L20  L21 L22 L23  L24
## 0tu.1       4  11 600   7  38  34 5709    4   143  123  242  190 170  84   40
## 0tu.10   1707 293   6 529  12   4 4272 4854 11342 2819 1265 2767 385 318 2221
## 0tu.100     0   0   0   2   0   0  196    3     0    0    4    1   4   0    2
## 0tu.1002    0   0   0   0   0   0    0    0     0    0    0    1   0   0    0
## 0tu.1007    0   0   0   0   0   0    0    0     0    0    0    0   0   0    0
## 0tu.101     0   7   0   0   0   0    0    0     0    0    0    0   0   0    0
##            L3   L4  L5   L6   L7 L8 L9  N1 N2 P1
## 0tu.1       2   14  14  227    7  5  5 382  4 88
## 0tu.10   2559 3423 197 1193 2133 29 56  17  3  9
## 0tu.100     0    0   3    0    1  0  0   0  0  0
## 0tu.1002    0    0   0    0    0  0  0   0  0  1
## 0tu.1007    0    0   0    0    0  0  0   0  0  0
## 0tu.101     0    0   0    0    0  0  0   0  0  0

Now convert to a matrix for Phyloseq

otumat <- as.matrix(import_table)
head(otumat)
##              A1   A10   A11   A12   A13   A15    A16 A17 A18 A19    A2 A20 A21
## 0tu.1    113349 78465 68476 18916 58723 42467 177164  16  16   1 57565 447 690
## 0tu.10      706   146   166     2  5135     4   7862  42  56   9    33  49  23
## 0tu.100     175     2     0     0     0     0    989   1   2   1     1   0   4
## 0tu.1002     23     0     0     0     0     0      0   0   0   0     1   0   0
## 0tu.1007      0     0     0     0     0     0     24   0   0   0     1   0   0
## 0tu.101       0     0     0     0     0     0      0   0   0   1     0   2   2
##          A22 A23 A24 A25  A26  A27   A28 A29    A3   A30   A31   A32   A33
## 0tu.1    153 247 102 247  782  224  4851  99 71375 58889 44823 49375 25282
## 0tu.10    54   9  26  13 1916 4303 12191  20   185  8566   178  6508   290
## 0tu.100    2   1   1   1  348  155   593   0     6     8    11   163    26
## 0tu.1002   0   0   0   0    0    0     0   0     0     0     1     0     0
## 0tu.1007   0   0   0   0    0    1     6   0     0     0     0     7     0
## 0tu.101    0   0   1   4    0   32     0   0     1     0     0     0     1
##            A34    A35    A4   A5    A6    A7    A8   A9 C1 C10 C11 C12 C13 C16
## 0tu.1     7776 184008 72355  618 56973 32874 34090 1433  3   3   1   0   5   6
## 0tu.10   15260    732    22 4119    48   376  7578 4754 22 184  67 439 634   0
## 0tu.100    717     20     0 1126     0     8   814  434  0   0   0   0   0   0
## 0tu.1002     0      0    13    0     0     0     0    0  0   0   0   0   0   0
## 0tu.1007     3      2     0   13     0     0     1    2  0   0   0   0   0   0
## 0tu.101      0      0     5    0     0     0     0   25  0   0   0   0   1   0
##          C17 C18 C19 C2 C20 C21 C22 C23 C24 C25 C26  C27  C28 C29  C3 C30 C31
## 0tu.1      9   7   2  0   4   2   0   0   0   9  10    4    7   1   4   7   1
## 0tu.10   197 776  35 97  59   8 106 170 111  58 117 1295 1556 260 139 448 252
## 0tu.100    0   0   0  1   0   0   0   0   0   0   5    0    1   0   0   0   0
## 0tu.1002   0   0   0  0   0   1   0   0   0   0   0    0    0   0   0   0   0
## 0tu.1007   0   0   0  0   0   0   0   0   0   0   0    0    0   0   0   0   0
## 0tu.101    0   0   0  0   0   0   0   1   1   1   0  262  431 275   0 245 725
##          C32 C33 C34  C35 C36  C37 C38 C39  C4 C40 C5 C6  C7 C8  C9  L1 L10
## 0tu.1      1  62   0    1   2    5   3   2   5   0  7  2   0  8   3   1   4
## 0tu.10   942  77 290  948 465 1498 401 316 233 516  7  8 105 68 223 432  26
## 0tu.100    0   0   0    0   0    0   0   0   0   0  0  0   0  0   0   0   0
## 0tu.1002   0   0   0    0   0    0   0   0   0   0  0  0   0  0   0   0   0
## 0tu.1007   0   0   0    0   0    0   0   0   0   0  0  0   0  0   0   0   0
## 0tu.101   54 264 385 1153 196  464 861 590   0 468  0  1   0  0   0   0   0
##           L11 L12 L13 L14 L15 L16  L17  L18   L19   L2  L20  L21 L22 L23  L24
## 0tu.1       4  11 600   7  38  34 5709    4   143  123  242  190 170  84   40
## 0tu.10   1707 293   6 529  12   4 4272 4854 11342 2819 1265 2767 385 318 2221
## 0tu.100     0   0   0   2   0   0  196    3     0    0    4    1   4   0    2
## 0tu.1002    0   0   0   0   0   0    0    0     0    0    0    1   0   0    0
## 0tu.1007    0   0   0   0   0   0    0    0     0    0    0    0   0   0    0
## 0tu.101     0   7   0   0   0   0    0    0     0    0    0    0   0   0    0
##            L3   L4  L5   L6   L7 L8 L9  N1 N2 P1
## 0tu.1       2   14  14  227    7  5  5 382  4 88
## 0tu.10   2559 3423 197 1193 2133 29 56  17  3  9
## 0tu.100     0    0   3    0    1  0  0   0  0  0
## 0tu.1002    0    0   0    0    0  0  0   0  0  1
## 0tu.1007    0    0   0    0    0  0  0   0  0  0
## 0tu.101     0    0   0    0    0  0  0   0  0  0

Now create an OTU object using the function otu_table

OTU_all <- otu_table(otumat, taxa_are_rows = TRUE)
head(OTU_all)
## OTU Table:          [6 taxa and 99 samples]
##                      taxa are rows
##              A1   A10   A11   A12   A13   A15    A16 A17 A18 A19    A2 A20 A21
## 0tu.1    113349 78465 68476 18916 58723 42467 177164  16  16   1 57565 447 690
## 0tu.10      706   146   166     2  5135     4   7862  42  56   9    33  49  23
## 0tu.100     175     2     0     0     0     0    989   1   2   1     1   0   4
## 0tu.1002     23     0     0     0     0     0      0   0   0   0     1   0   0
## 0tu.1007      0     0     0     0     0     0     24   0   0   0     1   0   0
## 0tu.101       0     0     0     0     0     0      0   0   0   1     0   2   2
##          A22 A23 A24 A25  A26  A27   A28 A29    A3   A30   A31   A32   A33
## 0tu.1    153 247 102 247  782  224  4851  99 71375 58889 44823 49375 25282
## 0tu.10    54   9  26  13 1916 4303 12191  20   185  8566   178  6508   290
## 0tu.100    2   1   1   1  348  155   593   0     6     8    11   163    26
## 0tu.1002   0   0   0   0    0    0     0   0     0     0     1     0     0
## 0tu.1007   0   0   0   0    0    1     6   0     0     0     0     7     0
## 0tu.101    0   0   1   4    0   32     0   0     1     0     0     0     1
##            A34    A35    A4   A5    A6    A7    A8   A9 C1 C10 C11 C12 C13 C16
## 0tu.1     7776 184008 72355  618 56973 32874 34090 1433  3   3   1   0   5   6
## 0tu.10   15260    732    22 4119    48   376  7578 4754 22 184  67 439 634   0
## 0tu.100    717     20     0 1126     0     8   814  434  0   0   0   0   0   0
## 0tu.1002     0      0    13    0     0     0     0    0  0   0   0   0   0   0
## 0tu.1007     3      2     0   13     0     0     1    2  0   0   0   0   0   0
## 0tu.101      0      0     5    0     0     0     0   25  0   0   0   0   1   0
##          C17 C18 C19 C2 C20 C21 C22 C23 C24 C25 C26  C27  C28 C29  C3 C30 C31
## 0tu.1      9   7   2  0   4   2   0   0   0   9  10    4    7   1   4   7   1
## 0tu.10   197 776  35 97  59   8 106 170 111  58 117 1295 1556 260 139 448 252
## 0tu.100    0   0   0  1   0   0   0   0   0   0   5    0    1   0   0   0   0
## 0tu.1002   0   0   0  0   0   1   0   0   0   0   0    0    0   0   0   0   0
## 0tu.1007   0   0   0  0   0   0   0   0   0   0   0    0    0   0   0   0   0
## 0tu.101    0   0   0  0   0   0   0   1   1   1   0  262  431 275   0 245 725
##          C32 C33 C34  C35 C36  C37 C38 C39  C4 C40 C5 C6  C7 C8  C9  L1 L10
## 0tu.1      1  62   0    1   2    5   3   2   5   0  7  2   0  8   3   1   4
## 0tu.10   942  77 290  948 465 1498 401 316 233 516  7  8 105 68 223 432  26
## 0tu.100    0   0   0    0   0    0   0   0   0   0  0  0   0  0   0   0   0
## 0tu.1002   0   0   0    0   0    0   0   0   0   0  0  0   0  0   0   0   0
## 0tu.1007   0   0   0    0   0    0   0   0   0   0  0  0   0  0   0   0   0
## 0tu.101   54 264 385 1153 196  464 861 590   0 468  0  1   0  0   0   0   0
##           L11 L12 L13 L14 L15 L16  L17  L18   L19   L2  L20  L21 L22 L23  L24
## 0tu.1       4  11 600   7  38  34 5709    4   143  123  242  190 170  84   40
## 0tu.10   1707 293   6 529  12   4 4272 4854 11342 2819 1265 2767 385 318 2221
## 0tu.100     0   0   0   2   0   0  196    3     0    0    4    1   4   0    2
## 0tu.1002    0   0   0   0   0   0    0    0     0    0    0    1   0   0    0
## 0tu.1007    0   0   0   0   0   0    0    0     0    0    0    0   0   0    0
## 0tu.101     0   7   0   0   0   0    0    0     0    0    0    0   0   0    0
##            L3   L4  L5   L6   L7 L8 L9  N1 N2 P1
## 0tu.1       2   14  14  227    7  5  5 382  4 88
## 0tu.10   2559 3423 197 1193 2133 29 56  17  3  9
## 0tu.100     0    0   3    0    1  0  0   0  0  0
## 0tu.1002    0    0   0    0    0  0  0   0  0  1
## 0tu.1007    0    0   0    0    0  0  0   0  0  0
## 0tu.101     0    0   0    0    0  0  0   0  0  0

The total number of sequences for each sample

colSums(OTU_all)
##     A1    A10    A11    A12    A13    A15    A16    A17    A18    A19     A2 
## 180344  83627  69685  65585  78971  48091 553080    649   1189    425  58335 
##    A20    A21    A22    A23    A24    A25    A26    A27    A28    A29     A3 
##   1351   1877    692   1008    890    980  59016  64992 220645    832  77216 
##    A30    A31    A32    A33    A34    A35     A4     A5     A6     A7     A8 
## 142596  54753 104439  67731 325583 331039 104809 219435  58010  59057 205737 
##     A9     C1    C10    C11    C12    C13    C16    C17    C18    C19     C2 
## 302729  36845  55258  74592  68346 153155    264  54963  75035  59585  46334 
##    C20    C21    C22    C23    C24    C25    C26    C27    C28    C29     C3 
##  66498  31614  44242  48813  48586  64641  62939  47603  50588  54901  71429 
##    C30    C31    C32    C33    C34    C35    C36    C37    C38    C39     C4 
##  50985  36004  42673  18624  51048  54782  47082  51279  47484  50040  43049 
##    C40     C5     C6     C7     C8     C9     L1    L10    L11    L12    L13 
##  45373  52582  64638  40623  35523  55198  59489  29140  38500  46667  10614 
##    L14    L15    L16    L17    L18    L19     L2    L20    L21    L22    L23 
##  48171 127935    173 127168  36745 148778 112775  74426  34030  27094  41554 
##    L24     L3     L4     L5     L6     L7     L8     L9     N1     N2     P1 
##  54327  49541  65420  51715 140335  54744  49721  56880   6317    470 399827

Looking in to the number of sequences into each samples: Samples for Argentine stem weevils: A17, A19, A22, A24, A25, A29, A18, A20, A21, A23 has sequences <2000 others samples have sequences `>1000

For CRW samples we need to remove C16, similarly L16 from lucerne weevil sample: both with <1000 So we decided to remove these samples before rarefaction, so that we don’t loose much information for other samples. Estimation of the abundance of differnet taxa within a sample are therefore relative, and it is necessary to normalize counts before comparing a single taxon between samples.

Before we proceed further, we will also be removing Negative, positive and parasite samples, to just get samples from weevils:

To get the subsample:

OTU <- subset(OTU_all, select = -c(A17,A19,A22,A24,A25,A29,C16,L16,A18,A20,A21,A23,N1,N2,P1,C13,C17,C18,C19,C20,C21,C33,L13,L17,A15,A35))

The total number of sequences for each sample from the subset

colSums(OTU)
##     A1    A10    A11    A12    A13    A16     A2    A26    A27    A28     A3 
## 180344  83627  69685  65585  78971 553080  58335  59016  64992 220645  77216 
##    A30    A31    A32    A33    A34     A4     A5     A6     A7     A8     A9 
## 142596  54753 104439  67731 325583 104809 219435  58010  59057 205737 302729 
##     C1    C10    C11    C12     C2    C22    C23    C24    C25    C26    C27 
##  36845  55258  74592  68346  46334  44242  48813  48586  64641  62939  47603 
##    C28    C29     C3    C30    C31    C32    C34    C35    C36    C37    C38 
##  50588  54901  71429  50985  36004  42673  51048  54782  47082  51279  47484 
##    C39     C4    C40     C5     C6     C7     C8     C9     L1    L10    L11 
##  50040  43049  45373  52582  64638  40623  35523  55198  59489  29140  38500 
##    L12    L14    L15    L18    L19     L2    L20    L21    L22    L23    L24 
##  46667  48171 127935  36745 148778 112775  74426  34030  27094  41554  54327 
##     L3     L4     L5     L6     L7     L8     L9 
##  49541  65420  51715 140335  54744  49721  56880

The sample with minimum number of sequences

min(colSums(OTU))
## [1] 27094

Plot a histogram of the number of sequences for each sample

hist(colSums(OTU), breaks = 20)

Now we will import the taxonomy table. This is the output from the Sintax analysis.

import_taxa <- read.table('raw_data/sintax_taxonomy.tsv',header=TRUE,sep='\t', row.names=1)
head(import_taxa)
##           Kingdom                    Phylum               Class
## 0tu.1    Bacteria            Proteobacteria Gammaproteobacteria
## 0tu.10   Bacteria            Actinobacteria      Actinobacteria
## 0tu.100  Bacteria Cyanobacteria/Chloroplast         Chloroplast
## 0tu.1002 Bacteria            Proteobacteria Alphaproteobacteria
## 0tu.1007 Bacteria            Actinobacteria      Actinobacteria
## 0tu.101  Bacteria            Proteobacteria Alphaproteobacteria
##                      Order              Family            Genus Confidence
## 0tu.1    Enterobacteriales  Enterobacteriaceae         Buchnera       0.27
## 0tu.10     Actinomycetales        Nocardiaceae      Rhodococcus       1.00
## 0tu.100        Chloroplast        Streptophyta                          NA
## 0tu.1002       Rhizobiales Methylobacteriaceae Methylobacterium       1.00
## 0tu.1007   Actinomycetales  Pseudonocardiaceae        Umezawaea       0.66
## 0tu.101        Rhizobiales        Brucellaceae     Ochrobactrum       1.00

Convert to a matrix

taxonomy <- as.matrix(import_taxa)

Create a taxonomy class object

TAX <- tax_table(taxonomy)

Import the sample metadata

metadata <- read.table('raw_data/sample_metadata_complete.tsv',header = T,sep='\t',row.names = 1)

metadata
##            Name Parasitization Sex     Site No.of.Parasites
## N1     Neg.qPCR              -   -        -              NA
## N2  Neg.Control              -   -        -              NA
## C1          Crw             NP   M   Rakaia              NA
## C2          Crw             NP   F   Rakaia              NA
## C3          Crw             NP   F   Rakaia              NA
## C4          Crw             NP   M   Rakaia              NA
## C5          Crw             NP   M   Rakaia              NA
## C6          Crw             NP   M   Rakaia              NA
## C7          Crw             NP   M   Rakaia              NA
## C8          Crw             NP   M   Rakaia              NA
## C9          Crw             NP   M   Rakaia              NA
## C10         Crw              P   M   Rakaia              NA
## C11         Crw              P   F   Rakaia              NA
## C12         Crw              P   F   Rakaia              NA
## C13     Par.Crw              -   -   Rakaia               1
## C16     Par.Crw              -   -   Rakaia               1
## C17     Par.Crw              -   -   Rakaia               1
## C18     Par.Crw              -   -   Rakaia               1
## C19     Par.Crw              -   -   Rakaia               1
## C20     Par.Crw              -   -   Rakaia               1
## C21     Par.Crw              -   -   Rakaia               4
## C22         Crw              P   F  Lincoln              NA
## C23         Crw              P   F  Lincoln              NA
## C24         Crw              P   F  Lincoln              NA
## C25         Crw              P   M  Lincoln              NA
## C26         Crw             NP   F  Lincoln              NA
## C27         Crw             NP   M  Lincoln              NA
## C28         Crw             NP   F  Lincoln              NA
## C29         Crw             NP   F  Lincoln              NA
## C30         Crw             NP   F  Lincoln              NA
## C31         Crw              P   F  Lincoln              NA
## C32         Crw              P   M  Lincoln              NA
## C33     Par.Crw              -   -  Lincoln               2
## C34         Crw             NP   M  Lincoln              NA
## C35         Crw              P   M  Lincoln              NA
## C36         Crw              P   F  Lincoln              NA
## C37         Crw              P   F  Lincoln              NA
## C38         Crw              P   M  Lincoln              NA
## C39         Crw              P   M  Lincoln              NA
## C40         Crw              P   F  Lincoln              NA
## P1  Pos.Control              -   -        -              NA
## L1           Lw             NP   F  Lincoln              NA
## L2           Lw             NP   M  Lincoln              NA
## L3           Lw             NP   F  Lincoln              NA
## L4           Lw             NP   F  Lincoln              NA
## L5           Lw              P   M  Lincoln              NA
## L6           Lw              P   M  Lincoln              NA
## L7           Lw             NP   F  Lincoln              NA
## L8           Lw              P   M  Lincoln              NA
## L9           Lw             NP   M  Lincoln              NA
## L10          Lw             NP   F  Lincoln              NA
## L11          Lw             NP   F  Lincoln              NA
## L12          Lw             NP   M  Lincoln              NA
## L13      Par.Lw              -   -  Lincoln               1
## L14          Lw              P   M  Lincoln              NA
## L15          Lw              P   M  Lincoln              NA
## L16          Lw              P   M  Lincoln              NA
## L17      Par.Lw              -   -  Lincoln               1
## L18          Lw             NP   F Grasmere              NA
## L19          Lw             NP   F Grasmere              NA
## L20          Lw             NP   F Grasmere              NA
## L21          Lw             NP   F Grasmere              NA
## L22          Lw             NP   M Grasmere              NA
## L23          Lw             NP   M Grasmere              NA
## L24          Lw              P   M Grasmere              NA
## A1          Asw             NP   M  Lincoln              NA
## A2          Asw             NP   M  Lincoln              NA
## A3          Asw             NP   M  Lincoln              NA
## A4          Asw              P   M  Lincoln              NA
## A5          Asw             NP   M  Lincoln              NA
## A6          Asw             NP   F  Lincoln              NA
## A7          Asw             NP   F  Lincoln              NA
## A8          Asw             NP   F  Lincoln              NA
## A9          Asw              P   F  Lincoln              NA
## A10         Asw             NP   F  Lincoln              NA
## A11         Asw             NP   F  Lincoln              NA
## A12         Asw              P   M  Lincoln              NA
## A13         Asw             NP   F  Lincoln              NA
## A15     Par.Asw              -   -  Lincoln               1
## A16         Asw              P   F  Lincoln              NA
## A17         Asw              P   M  Lincoln              NA
## A18         Asw              P   M  Lincoln              NA
## A19     Par.Asw              -   -  Lincoln               1
## A20     Par.Asw              -   -  Lincoln               2
## A21         Asw             NP   M  Lincoln              NA
## A22         Asw             NP   F  Lincoln              NA
## A23         Asw              P   M  Lincoln              NA
## A24         Asw              P   M  Lincoln              NA
## A25         Asw              P   F  Lincoln              NA
## A26         Asw              P   M  Lincoln              NA
## A27         Asw              P   M  Lincoln              NA
## A28         Asw              P   M  Lincoln              NA
## A29     Par.Asw              -   -  Lincoln               3
## A30         Asw             NP   M  Lincoln              NA
## A31         Asw             NP   F  Lincoln              NA
## A32         Asw              P   M  Lincoln              NA
## A33         Asw              P   -  Lincoln              NA
## A34         Asw              P   M  Lincoln              NA
## A35     Par.Asw              -   -  Lincoln               3

We need to remove the samples that we removed earlier from the OTU table, and we also need to remove column with No.of parasites

meta_data_weevils <- subset(metadata, select = -c(No.of.Parasites))
meta_data_weevils
##            Name Parasitization Sex     Site
## N1     Neg.qPCR              -   -        -
## N2  Neg.Control              -   -        -
## C1          Crw             NP   M   Rakaia
## C2          Crw             NP   F   Rakaia
## C3          Crw             NP   F   Rakaia
## C4          Crw             NP   M   Rakaia
## C5          Crw             NP   M   Rakaia
## C6          Crw             NP   M   Rakaia
## C7          Crw             NP   M   Rakaia
## C8          Crw             NP   M   Rakaia
## C9          Crw             NP   M   Rakaia
## C10         Crw              P   M   Rakaia
## C11         Crw              P   F   Rakaia
## C12         Crw              P   F   Rakaia
## C13     Par.Crw              -   -   Rakaia
## C16     Par.Crw              -   -   Rakaia
## C17     Par.Crw              -   -   Rakaia
## C18     Par.Crw              -   -   Rakaia
## C19     Par.Crw              -   -   Rakaia
## C20     Par.Crw              -   -   Rakaia
## C21     Par.Crw              -   -   Rakaia
## C22         Crw              P   F  Lincoln
## C23         Crw              P   F  Lincoln
## C24         Crw              P   F  Lincoln
## C25         Crw              P   M  Lincoln
## C26         Crw             NP   F  Lincoln
## C27         Crw             NP   M  Lincoln
## C28         Crw             NP   F  Lincoln
## C29         Crw             NP   F  Lincoln
## C30         Crw             NP   F  Lincoln
## C31         Crw              P   F  Lincoln
## C32         Crw              P   M  Lincoln
## C33     Par.Crw              -   -  Lincoln
## C34         Crw             NP   M  Lincoln
## C35         Crw              P   M  Lincoln
## C36         Crw              P   F  Lincoln
## C37         Crw              P   F  Lincoln
## C38         Crw              P   M  Lincoln
## C39         Crw              P   M  Lincoln
## C40         Crw              P   F  Lincoln
## P1  Pos.Control              -   -        -
## L1           Lw             NP   F  Lincoln
## L2           Lw             NP   M  Lincoln
## L3           Lw             NP   F  Lincoln
## L4           Lw             NP   F  Lincoln
## L5           Lw              P   M  Lincoln
## L6           Lw              P   M  Lincoln
## L7           Lw             NP   F  Lincoln
## L8           Lw              P   M  Lincoln
## L9           Lw             NP   M  Lincoln
## L10          Lw             NP   F  Lincoln
## L11          Lw             NP   F  Lincoln
## L12          Lw             NP   M  Lincoln
## L13      Par.Lw              -   -  Lincoln
## L14          Lw              P   M  Lincoln
## L15          Lw              P   M  Lincoln
## L16          Lw              P   M  Lincoln
## L17      Par.Lw              -   -  Lincoln
## L18          Lw             NP   F Grasmere
## L19          Lw             NP   F Grasmere
## L20          Lw             NP   F Grasmere
## L21          Lw             NP   F Grasmere
## L22          Lw             NP   M Grasmere
## L23          Lw             NP   M Grasmere
## L24          Lw              P   M Grasmere
## A1          Asw             NP   M  Lincoln
## A2          Asw             NP   M  Lincoln
## A3          Asw             NP   M  Lincoln
## A4          Asw              P   M  Lincoln
## A5          Asw             NP   M  Lincoln
## A6          Asw             NP   F  Lincoln
## A7          Asw             NP   F  Lincoln
## A8          Asw             NP   F  Lincoln
## A9          Asw              P   F  Lincoln
## A10         Asw             NP   F  Lincoln
## A11         Asw             NP   F  Lincoln
## A12         Asw              P   M  Lincoln
## A13         Asw             NP   F  Lincoln
## A15     Par.Asw              -   -  Lincoln
## A16         Asw              P   F  Lincoln
## A17         Asw              P   M  Lincoln
## A18         Asw              P   M  Lincoln
## A19     Par.Asw              -   -  Lincoln
## A20     Par.Asw              -   -  Lincoln
## A21         Asw             NP   M  Lincoln
## A22         Asw             NP   F  Lincoln
## A23         Asw              P   M  Lincoln
## A24         Asw              P   M  Lincoln
## A25         Asw              P   F  Lincoln
## A26         Asw              P   M  Lincoln
## A27         Asw              P   M  Lincoln
## A28         Asw              P   M  Lincoln
## A29     Par.Asw              -   -  Lincoln
## A30         Asw             NP   M  Lincoln
## A31         Asw             NP   F  Lincoln
## A32         Asw              P   M  Lincoln
## A33         Asw              P   -  Lincoln
## A34         Asw              P   M  Lincoln
## A35     Par.Asw              -   -  Lincoln
# making vector of row names that needs to be removed
row_names_to_remove <- c("A17","A19","A22","A24","A25","A29","C16","L16","A18","A20","A21","A23","N1","N2","P1","C13","C17","C18","C19","C20","C21","C33","L13","L17","A15","A35")

# removing the specified rows:
meta_data_weevils[!(row.names(meta_data_weevils) %in% row_names_to_remove), ]
##     Name Parasitization Sex     Site
## C1   Crw             NP   M   Rakaia
## C2   Crw             NP   F   Rakaia
## C3   Crw             NP   F   Rakaia
## C4   Crw             NP   M   Rakaia
## C5   Crw             NP   M   Rakaia
## C6   Crw             NP   M   Rakaia
## C7   Crw             NP   M   Rakaia
## C8   Crw             NP   M   Rakaia
## C9   Crw             NP   M   Rakaia
## C10  Crw              P   M   Rakaia
## C11  Crw              P   F   Rakaia
## C12  Crw              P   F   Rakaia
## C22  Crw              P   F  Lincoln
## C23  Crw              P   F  Lincoln
## C24  Crw              P   F  Lincoln
## C25  Crw              P   M  Lincoln
## C26  Crw             NP   F  Lincoln
## C27  Crw             NP   M  Lincoln
## C28  Crw             NP   F  Lincoln
## C29  Crw             NP   F  Lincoln
## C30  Crw             NP   F  Lincoln
## C31  Crw              P   F  Lincoln
## C32  Crw              P   M  Lincoln
## C34  Crw             NP   M  Lincoln
## C35  Crw              P   M  Lincoln
## C36  Crw              P   F  Lincoln
## C37  Crw              P   F  Lincoln
## C38  Crw              P   M  Lincoln
## C39  Crw              P   M  Lincoln
## C40  Crw              P   F  Lincoln
## L1    Lw             NP   F  Lincoln
## L2    Lw             NP   M  Lincoln
## L3    Lw             NP   F  Lincoln
## L4    Lw             NP   F  Lincoln
## L5    Lw              P   M  Lincoln
## L6    Lw              P   M  Lincoln
## L7    Lw             NP   F  Lincoln
## L8    Lw              P   M  Lincoln
## L9    Lw             NP   M  Lincoln
## L10   Lw             NP   F  Lincoln
## L11   Lw             NP   F  Lincoln
## L12   Lw             NP   M  Lincoln
## L14   Lw              P   M  Lincoln
## L15   Lw              P   M  Lincoln
## L18   Lw             NP   F Grasmere
## L19   Lw             NP   F Grasmere
## L20   Lw             NP   F Grasmere
## L21   Lw             NP   F Grasmere
## L22   Lw             NP   M Grasmere
## L23   Lw             NP   M Grasmere
## L24   Lw              P   M Grasmere
## A1   Asw             NP   M  Lincoln
## A2   Asw             NP   M  Lincoln
## A3   Asw             NP   M  Lincoln
## A4   Asw              P   M  Lincoln
## A5   Asw             NP   M  Lincoln
## A6   Asw             NP   F  Lincoln
## A7   Asw             NP   F  Lincoln
## A8   Asw             NP   F  Lincoln
## A9   Asw              P   F  Lincoln
## A10  Asw             NP   F  Lincoln
## A11  Asw             NP   F  Lincoln
## A12  Asw              P   M  Lincoln
## A13  Asw             NP   F  Lincoln
## A16  Asw              P   F  Lincoln
## A26  Asw              P   M  Lincoln
## A27  Asw              P   M  Lincoln
## A28  Asw              P   M  Lincoln
## A30  Asw             NP   M  Lincoln
## A31  Asw             NP   F  Lincoln
## A32  Asw              P   M  Lincoln
## A33  Asw              P   -  Lincoln
## A34  Asw              P   M  Lincoln

Create a Phyloseq sample_data-class

META <- sample_data(meta_data_weevils)

Create a Phyloseq object

Now that we have all the components, it is time to create a Phyloseq object

physeq <- phyloseq(OTU,TAX,META)
physeq
## phyloseq-class experiment-level object
## otu_table()   OTU Table:         [ 1142 taxa and 73 samples ]
## sample_data() Sample Data:       [ 73 samples by 4 sample variables ]
## tax_table()   Taxonomy Table:    [ 1142 taxa by 7 taxonomic ranks ]

If we had metadata variables as numerics, we need to convert them as characters, but in our case, we got them as characters, so no modifications needed.

Looking at the data

Now that we have our Phyloseq object, we will take a look at it. One of the first steps is to check alpha rarefaction of species richness. This is done to show that there has been sufficient sequencing to detect most species (OTUs).

rarecurve(t(otu_table(physeq)), step=50, cex=1)

create a bar plot of abundance

plot_bar(physeq, fill="Kingdom")

print(min(sample_sums(physeq)))
## [1] 27094
print(max(sample_sums(physeq)))
## [1] 553080

Rarefy the data

We can use rarefaction to simulate an even number of reads per sample. Rarefying the data is preferred for some analyses, though there is some debate. We will create a rarefied version of the Phyloseq object.

we will rarefy the data around 90% of the lowest sample

physeq.rarefied <- rarefy_even_depth(physeq, rngseed=1, sample.size=0.9*min(sample_sums(physeq)), replace=F)
## `set.seed(1)` was used to initialize repeatable random subsampling.
## Please record this for your records so others can reproduce.
## Try `set.seed(1); .Random.seed` for the full vector
## ...
## 20OTUs were removed because they are no longer 
## present in any sample after random subsampling
## ...

now plot the rerefied version

plot_bar(physeq.rarefied)

# Saving work to files You can save the Phyloseq object you just created, and then import it into another R session later. This way you do not have to re-import all the components separately.

Also, below are a couple of examples of saving graphs. There are many options for this that you can explore to create publication-quality graphics of your results

Save the phyloseq object

saveRDS(physeq, 'weevils_phyloseq.rds')

Also save the rarefied version

saveRDS(physeq.rarefied, 'weevils_phyloseq_rarefied.rds')

#Saving a graph to file

# open a pdf file
pdf('species_richness_plot.pdf')
# run the plot, or add the saved one
rarecurve(t(otu_table(physeq)), step=50, cex=1.5, col='blue',lty=2)
# close the pdf
dev.off()
## quartz_off_screen 
##                 2

Saving in jpeg

jpeg("species_richness_plot.jpg", width = 800, height = 800)
rarecurve(t(otu_table(physeq)), step=50, cex=1.5, col='blue',lty=2)
dev.off()
## quartz_off_screen 
##                 2

Resetting the graphics device, because the plot was not displayed properly

dev.off()
## null device 
##           1

Plotting taxonomy

plot_bar(physeq, fill='Phylum')

plot_bar(physeq.rarefied, fill='Phylum')

Now add ggplot facet_wrap tools to graph

barplot1 <- plot_bar(physeq.rarefied, fill='Phylum')
barplot1 +facet_wrap(~Name, scales="free_x", nrow = 1)

Combine visuals to present more information from non rarefied data

barplot2 <- plot_bar(physeq, x="Parasitization", fill = 'Phylum') + facet_wrap(~Name, scales = "free_x", nrow = 1)

barplot2

barplot3 <- plot_bar(physeq.rarefied, x="Parasitization", fill = 'Phylum') + facet_wrap(~Name, scales = "free_x", nrow = 1)

barplot3

barplot4 = plot_bar(physeq.rarefied, "Family", fill="Genus",facet_grid=~Name)
                     
barplot4+geom_point(aes(x=Family, y=Abundance), color="black", position = "jitter", size=3)

Community ecology

jac_dist <- distance(physeq.rarefied, method = "jaccard", binary = TRUE)
jac_dist
##            A1       A10       A11       A12       A13       A16        A2
## A10 0.7915408                                                            
## A11 0.8403909 0.7034483                                                  
## A12 0.9240506 0.7067669 0.6804124                                        
## A13 0.6746988 0.6467662 0.6588235 0.7954545                              
## A16 0.5991649 0.7549505 0.8379747 0.8841310 0.6788321                    
## A2  0.7313916 0.7415730 0.7500000 0.8450704 0.7488152 0.7956204          
## A26 0.6106719 0.7950450 0.8581395 0.9080460 0.7222222 0.4821092 0.8130631
## A27 0.6795096 0.7917526 0.8684760 0.9064449 0.7302231 0.5325342 0.8299595
## A28 0.7012448 0.8475336 0.9001513 0.9324324 0.7895522 0.5706447 0.8679525
## A3  0.5822785 0.7711599 0.8243243 0.8821549 0.6829268 0.6216216 0.7242525
## A30 0.6323851 0.7270195 0.8063584 0.8559078 0.6603774 0.5584158 0.7994652
## A31 0.6732892 0.7412791 0.8103976 0.8558282 0.6750700 0.5454545 0.8128492
## A32 0.6129707 0.7436548 0.8193717 0.8788660 0.6455696 0.4980392 0.7763819
## A33 0.6498674 0.6884615 0.7827869 0.8340249 0.6094891 0.5767442 0.7433962
## A34 0.6973501 0.8393939 0.8960245 0.9270517 0.7879699 0.5536960 0.8584337
## A4  0.7971831 0.6919192 0.7714286 0.7716049 0.6943231 0.7759815 0.7696078
## A5  0.6112150 0.7945493 0.8580645 0.8972163 0.7160752 0.4701627 0.8236515
## A6  0.8553459 0.6575342 0.6666667 0.6923077 0.7103825 0.8295739 0.7297297
## A7  0.6426513 0.6289593 0.7050000 0.7474227 0.5588235 0.6135266 0.7229437
## A8  0.6474501 0.7262248 0.8083832 0.8630952 0.6460674 0.5796813 0.7944444
## A9  0.6623377 0.7500000 0.8163265 0.8728324 0.6863271 0.5103306 0.7923497
## C1  0.7993921 0.7102273 0.7379310 0.8716216 0.7695853 0.8171021 0.7341040
## C10 0.6889535 0.7695652 0.7354497 0.8905473 0.6485356 0.7175926 0.7612613
## C11 0.8535826 0.7804878 0.7812500 0.8333333 0.7878788 0.8595642 0.8000000
## C12 0.6940299 0.7689655 0.8098859 0.9160584 0.6983607 0.6751055 0.7624113
## C2  0.7333333 0.7604563 0.7844828 0.8962656 0.7021277 0.7188841 0.7480315
## C22 0.7478006 0.7840376 0.7784091 0.8944444 0.7056277 0.7587822 0.7756098
## C23 0.7470238 0.7222222 0.7559524 0.8843931 0.6977778 0.7670588 0.7435897
## C24 0.7836991 0.7709497 0.6766917 0.8571429 0.6934673 0.7995110 0.7380952
## C25 0.7902736 0.7894737 0.7432432 0.8888889 0.7323944 0.7927711 0.7868852
## C26 0.7545455 0.7210526 0.7484277 0.8562500 0.6837209 0.7990654 0.7158470
## C27 0.7571429 0.7578947 0.8257576 0.9029851 0.7207792 0.7474950 0.7509025
## C28 0.7185355 0.7826087 0.8400000 0.9114754 0.7294118 0.7000000 0.7924528
## C29 0.7979003 0.8016529 0.8173077 0.9150943 0.7777778 0.8089172 0.8050847
## C3  0.7120743 0.7661692 0.7407407 0.8742515 0.6666667 0.7553957 0.7301587
## C30 0.7806122 0.7500000 0.8230088 0.9082969 0.7364621 0.7845188 0.7773279
## C31 0.7881137 0.7813765 0.8264840 0.9095023 0.7509158 0.7983193 0.7740586
## C32 0.7602180 0.7575758 0.7970297 0.9138756 0.7226562 0.7789934 0.7544643
## C34 0.7469880 0.7862069 0.8464419 0.9225092 0.7555556 0.7439516 0.7972028
## C35 0.7653061 0.7704280 0.8275862 0.8965517 0.7330961 0.7745303 0.8015564
## C36 0.8025316 0.7651822 0.8295964 0.9111111 0.7678571 0.7899160 0.8072289
## C37 0.7493857 0.7689531 0.8167331 0.9108527 0.7342193 0.7355372 0.7528090
## C38 0.7570332 0.7722008 0.8340426 0.9159664 0.7349823 0.7626050 0.7649402
## C39 0.7870130 0.7745902 0.8248848 0.9136364 0.7627737 0.7974684 0.7824268
## C4  0.7002584 0.7453184 0.8073770 0.9087302 0.7113402 0.7023555 0.8065693
## C40 0.7443038 0.7761194 0.8360656 0.9280000 0.7440273 0.7572614 0.7692308
## C5  0.9084967 0.8472222 0.7676768 0.8089888 0.8681319 0.9156328 0.8208955
## C6  0.8636364 0.8181818 0.6981132 0.8380952 0.8128342 0.8814815 0.8082192
## C7  0.7981651 0.7430168 0.7517241 0.8541667 0.7320574 0.8329412 0.7600000
## C8  0.7241379 0.7397260 0.7459459 0.8750000 0.6379310 0.7129412 0.7298578
## C9  0.7088235 0.7625571 0.7795699 0.8835979 0.6822034 0.7365967 0.7476190
## L1  0.7578348 0.7358491 0.7759563 0.8756757 0.7058824 0.7580645 0.7735849
## L10 0.7049180 0.7816456 0.8241379 0.8907850 0.7121212 0.6774194 0.7916667
## L11 0.6768868 0.7523810 0.8405316 0.8900000 0.6957831 0.6195426 0.7968750
## L12 0.7020057 0.7219731 0.7512953 0.8461538 0.6514523 0.7276888 0.7293578
## L14 0.6643836 0.7947977 0.8452012 0.9181818 0.7053824 0.6376238 0.8040936
## L15 0.7345133 0.8047337 0.8498403 0.9113924 0.7055394 0.6893204 0.8214286
## L18 0.6597222 0.7620482 0.8312102 0.8962264 0.6812865 0.6222222 0.7713415
## L19 0.6415094 0.7673716 0.8258065 0.8881789 0.6784661 0.6282828 0.7692308
## L2  0.6837782 0.7964377 0.8590426 0.9160105 0.7238806 0.6171004 0.8076923
## L20 0.6431818 0.7983193 0.8368580 0.8985075 0.6862745 0.6301370 0.8039773
## L21 0.6338384 0.7770492 0.8333333 0.8861210 0.6762821 0.6721649 0.7627119
## L22 0.6268344 0.7915633 0.8493506 0.9051282 0.7146341 0.5910781 0.7964824
## L23 0.6725441 0.7620690 0.8157895 0.9007353 0.6744186 0.6680761 0.7727273
## L24 0.6893939 0.7896552 0.8282443 0.9018868 0.6835017 0.6849894 0.7836879
## L3  0.6895787 0.8016997 0.8374233 0.9131737 0.7384196 0.6365422 0.8108883
## L4  0.6516588 0.7811550 0.8446602 0.9035370 0.6952663 0.6257669 0.7870370
## L5  0.6837782 0.8085642 0.8650794 0.9160105 0.7395577 0.6348624 0.8107417
## L6  0.7229730 0.7872340 0.8436482 0.8957655 0.7088235 0.6914062 0.8079268
## L7  0.7086420 0.7673611 0.8084291 0.8867925 0.6920530 0.6793249 0.7867133
## L8  0.7123656 0.7658730 0.8080357 0.9039301 0.7374101 0.7066667 0.7880000
## L9  0.7951070 0.7444444 0.7619048 0.8472222 0.7272727 0.8138425 0.7471264
##           A26       A27       A28        A3       A30       A31       A32
## A10                                                                      
## A11                                                                      
## A12                                                                      
## A13                                                                      
## A16                                                                      
## A2                                                                       
## A26                                                                      
## A27 0.5450082                                                            
## A28 0.5287671 0.4567901                                                  
## A3  0.6318898 0.6558559 0.6850282                                        
## A30 0.5468451 0.5543672 0.6050420 0.6409692                              
## A31 0.5736434 0.5425926 0.5974026 0.6430206 0.5410200                    
## A32 0.4857685 0.5157343 0.5578801 0.6122881 0.5000000 0.5408805          
## A33 0.6060606 0.6372549 0.7077827 0.6781003 0.6093366 0.6066838 0.5788235
## A34 0.5152778 0.4741144 0.3051948 0.6676259 0.6002825 0.6005789 0.5341880
## A4  0.8067941 0.7956778 0.8501441 0.7890173 0.7589744 0.7700535 0.7688679
## A5  0.4208633 0.5347003 0.4986523 0.6235955 0.5714286 0.5787546 0.5035714
## A6  0.8555046 0.8589212 0.8947368 0.8289474 0.8005698 0.8149254 0.8139535
## A7  0.6533333 0.6807229 0.7507418 0.6540698 0.5957447 0.6191781 0.5885287
## A8  0.5562016 0.5579710 0.6298883 0.6318182 0.5144766 0.5805740 0.5095137
## A9  0.5206287 0.5719361 0.5931721 0.6414254 0.5576923 0.5786026 0.5359343
## C1  0.8248337 0.8215010 0.8601190 0.7753165 0.7783784 0.7806268 0.7901235
## C10 0.7205240 0.7285429 0.7767857 0.6776119 0.7040816 0.6703297 0.6937799
## C11 0.8671171 0.8670757 0.9035608 0.8424437 0.8387978 0.8343023 0.8567901
## C12 0.6530612 0.6534091 0.6919708 0.6878173 0.6620690 0.6423358 0.6373626
## C2  0.7215447 0.6897881 0.7279412 0.7142857 0.6880952 0.6758794 0.7054945
## C22 0.7648352 0.7933071 0.8316252 0.7640118 0.7329843 0.7362637 0.7421687
## C23 0.7589286 0.7811245 0.8266667 0.7292308 0.7486911 0.7561644 0.7445255
## C24 0.8032037 0.8213552 0.8671642 0.7898089 0.7774725 0.7936963 0.7894737
## C25 0.8129176 0.8178138 0.8556548 0.7812500 0.7768817 0.7720798 0.8004866
## C26 0.7839644 0.8012048 0.8483063 0.7327044 0.7553191 0.7492958 0.7658537
## C27 0.7485714 0.7578947 0.7800546 0.7150000 0.7274725 0.7386364 0.7356557
## C28 0.7033582 0.7204117 0.7503356 0.7102804 0.7199170 0.6938326 0.7029703
## C29 0.8092369 0.8161765 0.8437936 0.7804878 0.7939110 0.8024390 0.7899344
## C3  0.7645740 0.7862903 0.8288690 0.7047619 0.7319035 0.7280453 0.7413793
## C30 0.7813121 0.7949183 0.8289655 0.7533156 0.7558140 0.7716346 0.7624190
## C31 0.7944112 0.7981651 0.8317107 0.7673797 0.7681499 0.7932692 0.7658643
## C32 0.7784679 0.7836812 0.8267045 0.7443820 0.7481663 0.7519182 0.7471526
## C34 0.7523810 0.7548501 0.7744154 0.7389163 0.7456522 0.7431818 0.7293388
## C35 0.7862745 0.7795993 0.8190608 0.7571802 0.7534562 0.7572115 0.7494600
## C36 0.7936508 0.7908257 0.8245125 0.7730871 0.7587822 0.7628362 0.7543860
## C37 0.7470817 0.7454874 0.7743733 0.7020725 0.7194570 0.7306792 0.7066381
## C38 0.7702970 0.7737226 0.8030726 0.7454068 0.7659091 0.7613365 0.7451404
## C39 0.8055556 0.8083942 0.8328691 0.7792553 0.8036530 0.7834550 0.7782609
## C4  0.7162978 0.6950758 0.7240876 0.6835106 0.6602871 0.6600000 0.6971678
## C40 0.7627451 0.7600000 0.7891061 0.7455013 0.7656250 0.7523585 0.7371795
## C5  0.9267735 0.9211618 0.9414414 0.8986486 0.9047619 0.8945783 0.9139241
## C6  0.8901602 0.8925620 0.9206587 0.8561873 0.8631285 0.8635015 0.8847118
## C7  0.8266667 0.8231707 0.8647845 0.7816456 0.7903226 0.7723343 0.7920792
## C8  0.7302632 0.7374749 0.7762763 0.6985075 0.6815789 0.6750000 0.7074341
## C9  0.7494553 0.7648221 0.8157895 0.7057057 0.7249357 0.6925208 0.7285714
## L1  0.7532751 0.7779961 0.8236152 0.7108434 0.7194805 0.7459893 0.7476415
## L10 0.6536204 0.6779964 0.7219917 0.6674817 0.6104784 0.6651481 0.6242038
## L11 0.6217822 0.6442831 0.6873239 0.6512195 0.6174497 0.6250000 0.6160338
## L12 0.7217391 0.7592233 0.8057971 0.6873156 0.6855670 0.7040000 0.6921241
## L14 0.6259542 0.6305506 0.6795580 0.6327014 0.5951860 0.6447368 0.6262626
## L15 0.6907407 0.6931034 0.7222222 0.6853147 0.6531049 0.6961207 0.6895874
## L18 0.6108949 0.6166365 0.6694678 0.6207729 0.5835189 0.6308725 0.5987526
## L19 0.6405354 0.6513274 0.6919890 0.6246973 0.6087912 0.6283784 0.6107660
## L2  0.6093190 0.5887372 0.6002766 0.6474359 0.5898990 0.6188525 0.6007605
## L20 0.6266417 0.6382609 0.6648276 0.6302326 0.6260504 0.6567164 0.6297030
## L21 0.6587302 0.6512059 0.7136812 0.5973684 0.6252874 0.6620047 0.6173913
## L22 0.5531136 0.6053068 0.5906593 0.5873362 0.5731463 0.6240000 0.5822306
## L23 0.6653226 0.6820702 0.7262411 0.6122995 0.6450116 0.6761229 0.6299559
## L24 0.6841046 0.6992620 0.7339972 0.6300268 0.6350711 0.7042254 0.6593886
## L3  0.6455224 0.6296296 0.6601671 0.6719818 0.6357895 0.6405229 0.6549020
## L4  0.6250000 0.6347197 0.6685552 0.6283619 0.5900901 0.6287016 0.6166667
## L5  0.6433566 0.6216667 0.6136986 0.6474359 0.6012024 0.6438632 0.5980952
## L6  0.6729679 0.6407942 0.7044199 0.6722090 0.6493506 0.6754967 0.6350515
## L7  0.6787149 0.6942910 0.7372881 0.6588542 0.6157518 0.6793349 0.6594360
## L8  0.7073684 0.7429112 0.7752161 0.6769663 0.7021792 0.7103275 0.7158837
## L9  0.8112360 0.8187373 0.8716814 0.7628205 0.7744565 0.7834758 0.7775000
##           A33       A34        A4        A5        A6        A7        A8
## A10                                                                      
## A11                                                                      
## A12                                                                      
## A13                                                                      
## A16                                                                      
## A2                                                                       
## A26                                                                      
## A27                                                                      
## A28                                                                      
## A3                                                                       
## A30                                                                      
## A31                                                                      
## A32                                                                      
## A33                                                                      
## A34 0.7056213                                                            
## A4  0.7397260 0.8440233                                                  
## A5  0.6069246 0.4911565 0.7935872                                        
## A6  0.7551020 0.8972810 0.7109827 0.8507463                              
## A7  0.5594406 0.7394578 0.7209302 0.6569647 0.7224880                    
## A8  0.6085859 0.6116643 0.7255435 0.5596330 0.7988166 0.5983607          
## A9  0.5592784 0.5759768 0.7610390 0.5177570 0.8171429 0.6180371 0.5745140
## C1  0.7647059 0.8641791 0.7962085 0.8295688 0.7181208 0.7372881 0.7724719
## C10 0.7062706 0.7878338 0.8122605 0.7504990 0.8125000 0.6590909 0.6711590
## C11 0.8470149 0.8995502 0.7925532 0.8804124 0.8029197 0.8008850 0.8418079
## C12 0.6798867 0.6877761 0.7886435 0.6842105 0.8284672 0.6749226 0.6563981
## C2  0.7205882 0.7445255 0.7827586 0.7281369 0.8306452 0.7316294 0.7053140
## C22 0.7651007 0.8199405 0.8033473 0.7838384 0.8074866 0.7058824 0.7324324
## C23 0.7253521 0.8271237 0.7826087 0.7886179 0.7740113 0.6987952 0.7349727
## C24 0.7584906 0.8627451 0.7951220 0.8270833 0.7397260 0.7123894 0.7749288
## C25 0.7761733 0.8545727 0.7990654 0.8209877 0.7875000 0.7458333 0.7945205
## C26 0.7561837 0.8420268 0.8053097 0.8089431 0.7530120 0.6970954 0.7486188
## C27 0.7486486 0.7802198 0.8056426 0.7531306 0.8134328 0.7681159 0.7438753
## C28 0.7251908 0.7520216 0.8163842 0.7105263 0.8441558 0.7418478 0.7299578
## C29 0.7872340 0.8410689 0.8388278 0.8079096 0.8294931 0.8006645 0.8123515
## C3  0.7340426 0.8258258 0.8146552 0.7738589 0.8089888 0.6708333 0.7382920
## C30 0.7566766 0.8294036 0.8257840 0.7996324 0.8034934 0.7827476 0.7793427
## C31 0.7797619 0.8337989 0.8115942 0.8051948 0.8274336 0.7922078 0.7862233
## C32 0.7468354 0.8271429 0.8195489 0.7884615 0.7990431 0.7526132 0.7667494
## C34 0.7574124 0.7779310 0.8302469 0.7500000 0.8296296 0.7879656 0.7702407
## C35 0.7500000 0.8227147 0.8090278 0.7886029 0.8135593 0.7596154 0.7710280
## C36 0.7823529 0.8199719 0.8226950 0.8000000 0.8097345 0.7870968 0.7767221
## C37 0.7500000 0.7693390 0.8167203 0.7540984 0.8223938 0.7515152 0.7391304
## C38 0.7658960 0.8067227 0.8184932 0.7690875 0.8298755 0.7843750 0.7720930
## C39 0.7964602 0.8365922 0.8315412 0.7977528 0.8153153 0.8064516 0.8079625
## C4  0.7055394 0.7332362 0.7933333 0.7279550 0.8274510 0.7111111 0.6707317
## C40 0.7549858 0.7893258 0.8322368 0.7642726 0.8458498 0.7945619 0.7744875
## C5  0.9087302 0.9377845 0.8713450 0.9284211 0.8073394 0.8605769 0.9069767
## C6  0.8498024 0.9151515 0.8125000 0.8924051 0.8032787 0.8009479 0.8470588
## C7  0.7898551 0.8637725 0.7766990 0.8312757 0.7565789 0.7510549 0.7746479
## C8  0.6949153 0.7837838 0.7530864 0.7520161 0.7817259 0.6653846 0.6864865
## C9  0.7205387 0.8109306 0.8221344 0.7695391 0.8071066 0.6896552 0.7342105
## L1  0.7348993 0.8136095 0.7759336 0.7948718 0.7979275 0.7110266 0.7253333
## L10 0.7135417 0.7110799 0.7515152 0.6787659 0.8169492 0.6820809 0.6065574
## L11 0.6613333 0.6851064 0.7790698 0.6470588 0.8446602 0.6550725 0.6295455
## L12 0.6778523 0.8095930 0.7160494 0.7514911 0.7733990 0.6666667 0.6693548
## L14 0.7201946 0.6624473 0.7934783 0.6452763 0.8727811 0.6775068 0.6219780
## L15 0.7395577 0.7185792 0.7614943 0.7076125 0.8606811 0.7333333 0.6767241
## L18 0.6632391 0.6633663 0.7933884 0.6487455 0.8390093 0.6648199 0.6169265
## L19 0.6743590 0.6952909 0.7427746 0.6541219 0.8190476 0.6544944 0.5986395
## L2  0.7066667 0.6243169 0.7745098 0.6262626 0.8619792 0.7169811 0.6004098
## L20 0.7098321 0.6512605 0.7675676 0.6573913 0.8508772 0.7012987 0.6017505
## L21 0.6937669 0.7152975 0.7692308 0.6910420 0.8498294 0.6746269 0.6378505
## L22 0.6636771 0.6013699 0.7905882 0.5664940 0.8524173 0.6808511 0.6143141
## L23 0.7247956 0.7169540 0.7628205 0.7000000 0.8505338 0.6687307 0.6289157
## L24 0.7042254 0.7247839 0.7884615 0.7266055 0.8419118 0.6934985 0.6481928
## L3  0.7272727 0.6652720 0.7903226 0.6466431 0.8684211 0.7300771 0.6474359
## L4  0.6855670 0.6699858 0.7736390 0.6373626 0.8485804 0.6514286 0.6050228
## L5  0.7180617 0.6110345 0.7864078 0.6600985 0.8708010 0.7109005 0.5975359
## L6  0.7363184 0.6987448 0.7652174 0.6890459 0.8512658 0.7052342 0.6252822
## L7  0.7182320 0.7354196 0.7434211 0.6904315 0.8272059 0.6645768 0.6286408
## L8  0.7425150 0.7753623 0.7797834 0.7251462 0.8396624 0.7006803 0.6909548
## L9  0.7683824 0.8639761 0.7894737 0.8316222 0.7417219 0.7307692 0.7718310
##            A9        C1       C10       C11       C12        C2       C22
## A10                                                                      
## A11                                                                      
## A12                                                                      
## A13                                                                      
## A16                                                                      
## A2                                                                       
## A26                                                                      
## A27                                                                      
## A28                                                                      
## A3                                                                       
## A30                                                                      
## A31                                                                      
## A32                                                                      
## A33                                                                      
## A34                                                                      
## A4                                                                       
## A5                                                                       
## A6                                                                       
## A7                                                                       
## A8                                                                       
## A9                                                                       
## C1  0.7972973                                                            
## C10 0.7186701 0.6698565                                                  
## C11 0.8453039 0.7337662 0.7635468                                        
## C12 0.6565421 0.7200000 0.6490066 0.7642586                              
## C2  0.7109005 0.6844262 0.6389892 0.7594937 0.6656805                    
## C22 0.7831633 0.7609756 0.6283186 0.8052632 0.7114094 0.6828358          
## C23 0.7406417 0.6878307 0.6367713 0.7722222 0.7142857 0.6806084 0.6854460
## C24 0.7967033 0.6728395 0.6780488 0.7297297 0.7454545 0.7183673 0.6720430
## C25 0.7924528 0.6764706 0.6682464 0.7547170 0.7455830 0.6987952 0.7213930
## C26 0.7706667 0.6994536 0.6513761 0.7588235 0.7177700 0.6784314 0.6567164
## C27 0.7400881 0.7400722 0.7101911 0.8074074 0.7493473 0.6341463 0.7508197
## C28 0.7211740 0.7714286 0.7270115 0.8424437 0.7512077 0.6611570 0.7748538
## C29 0.7990544 0.7608696 0.7689531 0.8482143 0.7900875 0.6983051 0.7713178
## C3  0.7606383 0.6721311 0.5693780 0.7218935 0.6866197 0.6588235 0.6519608
## C30 0.7834101 0.7291667 0.7377622 0.8119658 0.7677054 0.6797386 0.7518519
## C31 0.7958237 0.7510549 0.7473310 0.8354978 0.7828571 0.6963696 0.7806691
## C32 0.7682927 0.7064220 0.6745098 0.7740385 0.7439024 0.6537102 0.7183673
## C34 0.7635575 0.7824561 0.7406250 0.8530466 0.7706186 0.6843658 0.7929936
## C35 0.7808219 0.7510040 0.7301038 0.8360656 0.7715877 0.6688312 0.7527273
## C36 0.7865429 0.7341772 0.7464789 0.8181818 0.7784091 0.6788079 0.7749077
## C37 0.7522321 0.7175573 0.7147541 0.8069498 0.7197802 0.6406250 0.7525424
## C38 0.7592593 0.7628458 0.7275862 0.8326531 0.7694444 0.6535948 0.7634409
## C39 0.8032407 0.7489362 0.7364621 0.8185841 0.7920228 0.6632653 0.7651515
## C4  0.7199074 0.6693548 0.6263345 0.7782258 0.6510264 0.6205788 0.7071429
## C40 0.7619048 0.7576923 0.7324415 0.8300395 0.7656676 0.6254072 0.7543860
## C5  0.9059829 0.7894737 0.8270270 0.7476636 0.8764479 0.8235294 0.8443114
## C6  0.8700565 0.7535211 0.7606383 0.7500000 0.8282443 0.7894737 0.8125000
## C7  0.8123324 0.6585366 0.6730769 0.6959459 0.7272727 0.7137097 0.7208122
## C8  0.7202073 0.6030928 0.6085106 0.6810811 0.6498316 0.6037736 0.6939655
## C9  0.7429306 0.6831683 0.6000000 0.7486911 0.6621622 0.6630037 0.6929825
## L1  0.7602041 0.7476190 0.7032520 0.7578947 0.7156863 0.7342657 0.7552743
## L10 0.6805252 0.7581699 0.7176471 0.8193980 0.6684073 0.7018970 0.7515152
## L11 0.6541850 0.7912773 0.7204611 0.8354839 0.6649485 0.6978610 0.7647059
## L12 0.7265823 0.7214612 0.6823529 0.8009479 0.6942675 0.7281879 0.7213115
## L14 0.6546610 0.7810651 0.7247956 0.8473054 0.6908213 0.7095960 0.7666667
## L15 0.6877637 0.8053892 0.7534247 0.8038585 0.7222222 0.7455919 0.8060942
## L18 0.6296296 0.7659574 0.6985915 0.8409786 0.6566416 0.6745407 0.7304348
## L19 0.6361656 0.7638037 0.6772334 0.8286604 0.6506329 0.6892950 0.7606838
## L2  0.6207585 0.7844156 0.6997519 0.8364116 0.6561798 0.6595745 0.7804878
## L20 0.6687243 0.7780980 0.6885246 0.8323529 0.6746411 0.6859296 0.7608696
## L21 0.6924779 0.7440273 0.6539683 0.8401361 0.6929134 0.6657143 0.7460815
## L22 0.6176471 0.7980050 0.7098321 0.8596491 0.6578947 0.6943820 0.7732697
## L23 0.6945701 0.7578947 0.6601307 0.8362989 0.6883469 0.7020057 0.7508091
## L24 0.6979405 0.7598566 0.6732673 0.8315018 0.6958904 0.6988304 0.7315436
## L3  0.6864754 0.8091168 0.7154472 0.8258258 0.6699029 0.6606218 0.7671233
## L4  0.6395604 0.7777778 0.7011494 0.8173077 0.6443299 0.6939314 0.7485380
## L5  0.6608527 0.7906977 0.7373494 0.8455497 0.6681514 0.6965517 0.7893462
## L6  0.6844350 0.7648903 0.7359551 0.8199357 0.6834171 0.6992084 0.7863248
## L7  0.6766744 0.7544484 0.7028754 0.7947761 0.6740331 0.6794118 0.7434211
## L8  0.7283654 0.7611336 0.7163121 0.8127660 0.7023810 0.6977492 0.7388060
## L9  0.8096515 0.6686747 0.7235023 0.7798742 0.7553191 0.7548638 0.7836538
##           C23       C24       C25       C26       C27       C28       C29
## A10                                                                      
## A11                                                                      
## A12                                                                      
## A13                                                                      
## A16                                                                      
## A2                                                                       
## A26                                                                      
## A27                                                                      
## A28                                                                      
## A3                                                                       
## A30                                                                      
## A31                                                                      
## A32                                                                      
## A33                                                                      
## A34                                                                      
## A4                                                                       
## A5                                                                       
## A6                                                                       
## A7                                                                       
## A8                                                                       
## A9                                                                       
## C1                                                                       
## C10                                                                      
## C11                                                                      
## C12                                                                      
## C2                                                                       
## C22                                                                      
## C23                                                                      
## C24 0.6611111                                                            
## C25 0.7258883 0.6543210                                                  
## C26 0.5957447 0.6250000 0.6902174                                        
## C27 0.7068966 0.7785714 0.7649123 0.7370242                              
## C28 0.7292308 0.7859425 0.7888199 0.7484472 0.3937500                    
## C29 0.7357724 0.7860262 0.7899160 0.7613169 0.4452830 0.4641638          
## C3  0.6748768 0.6279070 0.6702703 0.6296296 0.7285223 0.7289720 0.7407407
## C30 0.7070312 0.7478992 0.7479675 0.6951220 0.3698113 0.4488449 0.3788546
## C31 0.7470817 0.7702128 0.7795918 0.7213115 0.3916350 0.4633333 0.3901345
## C32 0.7112971 0.7327189 0.7333333 0.6929825 0.4448669 0.4898649 0.4318182
## C34 0.7525084 0.8153310 0.7762238 0.7736486 0.4252492 0.4246154 0.4351145
## C35 0.7330827 0.7642276 0.7588933 0.7176471 0.4357143 0.4133333 0.3739130
## C36 0.7315175 0.7637131 0.7632653 0.7509881 0.3947368 0.4703947 0.4217391
## C37 0.7117438 0.7769517 0.7490775 0.7292419 0.3523132 0.4294671 0.4166667
## C38 0.7303371 0.7952756 0.7509881 0.7442748 0.3941606 0.4066667 0.4000000
## C39 0.7351779 0.7787234 0.7727273 0.7398374 0.4053030 0.4493243 0.3698630
## C4  0.6911765 0.7283465 0.7386364 0.6743295 0.7053824 0.6973684 0.7322581
## C40 0.7216117 0.7984791 0.7835821 0.7490775 0.4042553 0.4358974 0.4180328
## C5  0.8238994 0.8153846 0.8201439 0.7959184 0.8750000 0.9096990 0.8965517
## C6  0.7546012 0.7022901 0.7412587 0.7385621 0.8352490 0.8445946 0.8177340
## C7  0.6483516 0.6932515 0.6959064 0.6514286 0.7242647 0.7620579 0.7695652
## C8  0.6313364 0.6733668 0.6763285 0.6651163 0.6821192 0.6867470 0.7720588
## C9  0.6666667 0.6717949 0.6813725 0.6243902 0.6856187 0.6614907 0.6944444
## L1  0.7379913 0.7326733 0.7570093 0.7488789 0.7539936 0.7665706 0.7881041
## L10 0.7469136 0.8006431 0.7993730 0.7663551 0.7512315 0.7471264 0.7863014
## L11 0.7567568 0.7981073 0.8006135 0.7757576 0.6862245 0.6988235 0.7744565
## L12 0.6923077 0.7122642 0.7252252 0.6844444 0.7476923 0.7393768 0.7706093
## L14 0.7696629 0.7910448 0.8005780 0.7806268 0.7347418 0.7206208 0.8005051
## L15 0.7681159 0.8012232 0.8176471 0.7971014 0.7738928 0.7712418 0.8333333
## L18 0.7657143 0.7723077 0.7606061 0.7250755 0.7094431 0.6993166 0.7754569
## L19 0.7345133 0.7739938 0.7771084 0.7303030 0.6945813 0.6912442 0.7671958
## L2  0.7493671 0.8056995 0.7829457 0.7746835 0.7121212 0.6839917 0.7708333
## L20 0.7671233 0.8017241 0.8005618 0.7535411 0.7420091 0.7306034 0.7945545
## L21 0.7371795 0.7845118 0.7674419 0.7326733 0.7404580 0.7279236 0.7859155
## L22 0.7759036 0.8154613 0.8054187 0.7823961 0.7232704 0.7034068 0.7942478
## L23 0.7458746 0.7651246 0.7689655 0.7372014 0.7538860 0.7341463 0.7877907
## L24 0.7641196 0.7717391 0.7667845 0.7386760 0.7585302 0.7413793 0.7899408
## L3  0.7597765 0.7947214 0.7971429 0.7600000 0.7206573 0.6928251 0.7634961
## L4  0.7212121 0.7805643 0.7723077 0.7477204 0.7336562 0.7073733 0.7857143
## L5  0.7650000 0.8149100 0.8136020 0.7839196 0.7473684 0.7263581 0.8013544
## L6  0.7500000 0.8018576 0.8006042 0.7500000 0.7445783 0.7324263 0.7845745
## L7  0.7382550 0.7706093 0.7785467 0.7379310 0.7480315 0.7346437 0.7817109
## L8  0.7328244 0.7593361 0.7640000 0.7272727 0.7663818 0.7605263 0.7781457
## L9  0.7650000 0.7337278 0.7272727 0.7393617 0.7703180 0.7784810 0.7543860
##            C3       C30       C31       C32       C34       C35       C36
## A10                                                                      
## A11                                                                      
## A12                                                                      
## A13                                                                      
## A16                                                                      
## A2                                                                       
## A26                                                                      
## A27                                                                      
## A28                                                                      
## A3                                                                       
## A30                                                                      
## A31                                                                      
## A32                                                                      
## A33                                                                      
## A34                                                                      
## A4                                                                       
## A5                                                                       
## A6                                                                       
## A7                                                                       
## A8                                                                       
## A9                                                                       
## C1                                                                       
## C10                                                                      
## C11                                                                      
## C12                                                                      
## C2                                                                       
## C22                                                                      
## C23                                                                      
## C24                                                                      
## C25                                                                      
## C26                                                                      
## C27                                                                      
## C28                                                                      
## C29                                                                      
## C3                                                                       
## C30 0.7011952                                                            
## C31 0.7269076 0.3521739                                                  
## C32 0.6651982 0.4533898 0.4247788                                        
## C34 0.7397260 0.4363636 0.4291045 0.4869888                              
## C35 0.7181467 0.3983740 0.3617021 0.4411765 0.4593640                    
## C36 0.7160000 0.3702128 0.3879310 0.4482759 0.4432234 0.4314516          
## C37 0.7065217 0.4128788 0.3984375 0.4409449 0.4425676 0.4259259 0.3828125
## C38 0.7300380 0.3902439 0.3941909 0.4393305 0.4130435 0.3984064 0.4105691
## C39 0.7037037 0.3819742 0.3423423 0.4675325 0.4307116 0.3771186 0.3612335
## C4  0.6804511 0.7173913 0.7335423 0.7086093 0.7290503 0.7147239 0.7210031
## C40 0.7259259 0.4263566 0.3927126 0.4801587 0.4225352 0.4153846 0.4335938
## C5  0.8104575 0.8755760 0.8809524 0.8724490 0.9125475 0.8888889 0.9086758
## C6  0.7307692 0.8073394 0.8433180 0.8150000 0.8814815 0.8434783 0.8302752
## C7  0.6228571 0.7375000 0.7489362 0.7096774 0.7852113 0.7640000 0.7372881
## C8  0.6095238 0.6985294 0.7222222 0.6865079 0.7388535 0.7234043 0.6928839
## C9  0.5650000 0.6525097 0.6821705 0.6186441 0.7052980 0.6501901 0.6870229
## L1  0.7031963 0.7773852 0.7472119 0.7382812 0.7875000 0.7649123 0.7689531
## L10 0.7310127 0.7653333 0.7828418 0.7675070 0.7592138 0.7786458 0.7754011
## L11 0.7298137 0.7506631 0.7680000 0.7308782 0.7270471 0.7545692 0.7540107
## L12 0.6740088 0.7568493 0.7622378 0.7072243 0.7764350 0.7491525 0.7697595
## L14 0.7376093 0.7807882 0.7880299 0.7708333 0.7655172 0.7869249 0.7688442
## L15 0.7861272 0.8098765 0.8292079 0.8200514 0.8063781 0.8240964 0.8163772
## L18 0.7177177 0.7493606 0.7596899 0.7405405 0.7204819 0.7436548 0.7525773
## L19 0.7033639 0.7343750 0.7447368 0.7315068 0.7184466 0.7448980 0.7441253
## L2  0.7493606 0.7590090 0.7511521 0.7488152 0.7136659 0.7312073 0.7534247
## L20 0.7321937 0.7603912 0.7733990 0.7525773 0.7291667 0.7668269 0.7815534
## L21 0.7119205 0.7643836 0.7653631 0.7630058 0.7518987 0.7513661 0.7814208
## L22 0.7425000 0.7500000 0.7699115 0.7540230 0.7406639 0.7693966 0.7746171
## L23 0.6888112 0.7690141 0.7630058 0.7416413 0.7427822 0.7591036 0.7727273
## L24 0.7077465 0.7672414 0.7861272 0.7507692 0.7473404 0.7642045 0.7851003
## L3  0.7457143 0.7629630 0.7480916 0.7486911 0.7071429 0.7356608 0.7568922
## L4  0.7009346 0.7686375 0.7792208 0.7506849 0.7415459 0.7595908 0.7877238
## L5  0.7620253 0.7890110 0.7820225 0.7862069 0.7489451 0.7704194 0.7893570
## L6  0.7574850 0.7674419 0.7716535 0.7629428 0.7553957 0.7709924 0.7835052
## L7  0.6936620 0.7449275 0.7710145 0.7575758 0.7726098 0.7563739 0.7594203
## L8  0.7023810 0.7805643 0.7581699 0.7465753 0.7650430 0.7732919 0.7612903
## L9  0.6629834 0.7489712 0.7500000 0.7110092 0.7816901 0.7550201 0.7541667
##           C37       C38       C39        C4       C40        C5        C6
## A10                                                                      
## A11                                                                      
## A12                                                                      
## A13                                                                      
## A16                                                                      
## A2                                                                       
## A26                                                                      
## A27                                                                      
## A28                                                                      
## A3                                                                       
## A30                                                                      
## A31                                                                      
## A32                                                                      
## A33                                                                      
## A34                                                                      
## A4                                                                       
## A5                                                                       
## A6                                                                       
## A7                                                                       
## A8                                                                       
## A9                                                                       
## C1                                                                       
## C10                                                                      
## C11                                                                      
## C12                                                                      
## C2                                                                       
## C22                                                                      
## C23                                                                      
## C24                                                                      
## C25                                                                      
## C26                                                                      
## C27                                                                      
## C28                                                                      
## C29                                                                      
## C3                                                                       
## C30                                                                      
## C31                                                                      
## C32                                                                      
## C34                                                                      
## C35                                                                      
## C36                                                                      
## C37                                                                      
## C38 0.3576923                                                            
## C39 0.4000000 0.3039648                                                  
## C4  0.6863905 0.7280967 0.7398119                                        
## C40 0.3992674 0.3032787 0.3333333 0.7095808                              
## C5  0.8911290 0.8995633 0.8851675 0.8454936 0.9083333                    
## C6  0.8266129 0.8448276 0.8142857 0.8125000 0.8416667 0.7391304          
## C7  0.7396226 0.7560000 0.7413793 0.7086614 0.7748092 0.7280000 0.7226277
## C8  0.6815068 0.7298246 0.7296296 0.5858209 0.7172414 0.8268156 0.7914439
## C9  0.6805556 0.6923077 0.6533865 0.6784452 0.6750903 0.8352273 0.7514124
## L1  0.7475083 0.7793103 0.7728938 0.7162630 0.7661017 0.8715084 0.8152174
## L10 0.7525253 0.7766234 0.7783784 0.6986667 0.7634961 0.8961938 0.8605442
## L11 0.7128205 0.7328042 0.7500000 0.6981627 0.7198953 0.9063545 0.8679868
## L12 0.7207792 0.7466216 0.7605634 0.7278689 0.7629870 0.8652850 0.7886598
## L14 0.7201946 0.7580247 0.7930175 0.7000000 0.7578692 0.9133127 0.8707692
## L15 0.7843602 0.8106796 0.8283582 0.7481481 0.8095238 0.9061489 0.8726115
## L18 0.6934673 0.7353690 0.7552083 0.6854220 0.7131980 0.9116719 0.8647799
## L19 0.7042607 0.7397959 0.7400531 0.6930946 0.7240506 0.9073482 0.8262295
## L2  0.7008929 0.7295455 0.7528868 0.6697460 0.7270694 0.9029650 0.8750000
## L20 0.7159905 0.7649880 0.7783251 0.7024390 0.7529691 0.9066265 0.8618619
## L21 0.7146667 0.7527174 0.7743733 0.6994536 0.7426273 0.8916968 0.8586572
## L22 0.7099567 0.7434211 0.7770419 0.6946903 0.7326087 0.9121447 0.8708010
## L23 0.7107438 0.7569832 0.7723343 0.7022472 0.7465565 0.8943396 0.8339623
## L24 0.7397260 0.7655367 0.7884058 0.7138810 0.7753425 0.8910506 0.8196078
## L3  0.7087379 0.7369727 0.7305699 0.6817043 0.7121588 0.8947368 0.8523077
## L4  0.7218045 0.7480720 0.7780679 0.6736842 0.7449495 0.8907285 0.8603896
## L5  0.7180617 0.7605322 0.7919463 0.6819222 0.7576419 0.9088472 0.8750000
## L6  0.7300000 0.7627551 0.7671958 0.6961039 0.7593985 0.8862876 0.8631922
## L7  0.7252747 0.7647059 0.7803468 0.6914286 0.7575758 0.8798450 0.8314176
## L8  0.7349398 0.7822086 0.7647059 0.7182663 0.7629179 0.8761468 0.8348214
## L9  0.7500000 0.7716535 0.7531915 0.7859779 0.7803030 0.8489209 0.7862069
##            C7        C8        C9        L1       L10       L11       L12
## A10                                                                      
## A11                                                                      
## A12                                                                      
## A13                                                                      
## A16                                                                      
## A2                                                                       
## A26                                                                      
## A27                                                                      
## A28                                                                      
## A3                                                                       
## A30                                                                      
## A31                                                                      
## A32                                                                      
## A33                                                                      
## A34                                                                      
## A4                                                                       
## A5                                                                       
## A6                                                                       
## A7                                                                       
## A8                                                                       
## A9                                                                       
## C1                                                                       
## C10                                                                      
## C11                                                                      
## C12                                                                      
## C2                                                                       
## C22                                                                      
## C23                                                                      
## C24                                                                      
## C25                                                                      
## C26                                                                      
## C27                                                                      
## C28                                                                      
## C29                                                                      
## C3                                                                       
## C30                                                                      
## C31                                                                      
## C32                                                                      
## C34                                                                      
## C35                                                                      
## C36                                                                      
## C37                                                                      
## C38                                                                      
## C39                                                                      
## C4                                                                       
## C40                                                                      
## C5                                                                       
## C6                                                                       
## C7                                                                       
## C8  0.6414141                                                            
## C9  0.6800000 0.6244541                                                  
## L1  0.7746479 0.7053942 0.6936170                                        
## L10 0.7923323 0.6960486 0.7073171 0.6331169                              
## L11 0.8012422 0.6955224 0.6909091 0.6509434 0.5122616                    
## L12 0.7305936 0.6733871 0.6556017 0.6223176 0.5779221 0.6105919          
## L14 0.8285714 0.7050562 0.7298050 0.6557864 0.5233161 0.4869110 0.6297376
## L15 0.8254438 0.7344633 0.7660167 0.6826347 0.5494792 0.5400517 0.6276276
## L18 0.7904192 0.6811594 0.7106017 0.6891496 0.5463918 0.5445293 0.6312684
## L19 0.7507788 0.6744868 0.6891496 0.7130435 0.5503876 0.5594937 0.6156156
## L2  0.7896104 0.6911392 0.7037975 0.6605263 0.5609195 0.5324074 0.6615776
## L20 0.8146067 0.7076503 0.7107438 0.7011173 0.5907990 0.6018957 0.6758242
## L21 0.7635135 0.6719745 0.7084639 0.7138365 0.6010638 0.6057441 0.6433121
## L22 0.8118812 0.6921182 0.7201946 0.7336562 0.6056645 0.5943601 0.6829268
## L23 0.7605634 0.6915584 0.6907895 0.6789298 0.5658263 0.5907859 0.6174497
## L24 0.7887324 0.7091503 0.7128713 0.6836735 0.5882353 0.6419098 0.6442953
## L3  0.7692308 0.7027778 0.6875000 0.6578947 0.5671642 0.5721271 0.6740947
## L4  0.7801858 0.6873156 0.6865672 0.6170886 0.5027027 0.4560440 0.5900621
## L5  0.8112245 0.7202970 0.7450980 0.6462766 0.5235849 0.5117371 0.6547315
## L6  0.7901235 0.7159420 0.7304348 0.6697248 0.5040650 0.5224274 0.6303030
## L7  0.7832168 0.6474576 0.6866667 0.6134752 0.5146199 0.5546218 0.5833333
## L8  0.7692308 0.6996337 0.6742424 0.6120000 0.5545171 0.6158358 0.6000000
## L9  0.7413793 0.7142857 0.6616162 0.6063830 0.6794425 0.6969697 0.6188119
##           L14       L15       L18       L19        L2       L20       L21
## A10                                                                      
## A11                                                                      
## A12                                                                      
## A13                                                                      
## A16                                                                      
## A2                                                                       
## A26                                                                      
## A27                                                                      
## A28                                                                      
## A3                                                                       
## A30                                                                      
## A31                                                                      
## A32                                                                      
## A33                                                                      
## A34                                                                      
## A4                                                                       
## A5                                                                       
## A6                                                                       
## A7                                                                       
## A8                                                                       
## A9                                                                       
## C1                                                                       
## C10                                                                      
## C11                                                                      
## C12                                                                      
## C2                                                                       
## C22                                                                      
## C23                                                                      
## C24                                                                      
## C25                                                                      
## C26                                                                      
## C27                                                                      
## C28                                                                      
## C29                                                                      
## C3                                                                       
## C30                                                                      
## C31                                                                      
## C32                                                                      
## C34                                                                      
## C35                                                                      
## C36                                                                      
## C37                                                                      
## C38                                                                      
## C39                                                                      
## C4                                                                       
## C40                                                                      
## C5                                                                       
## C6                                                                       
## C7                                                                       
## C8                                                                       
## C9                                                                       
## L1                                                                       
## L10                                                                      
## L11                                                                      
## L12                                                                      
## L14                                                                      
## L15 0.5088608                                                            
## L18 0.5463415 0.5985577                                                  
## L19 0.5847255 0.6024096 0.5176768                                        
## L2  0.5180180 0.5792952 0.5826087 0.5860566                              
## L20 0.5917431 0.6278539 0.5024631 0.5421687 0.5653105                    
## L21 0.5581395 0.6442786 0.4918033 0.5514512 0.6294643 0.5194805          
## L22 0.5911950 0.6299376 0.5265487 0.5331858 0.5808967 0.4900662 0.5462963
## L23 0.5796345 0.6135770 0.5138122 0.5138889 0.5915493 0.5173333 0.4984985
## L24 0.5857520 0.6520619 0.5399449 0.5718157 0.6545455 0.5189189 0.5400593
## L3  0.5558195 0.6353211 0.5967366 0.5938967 0.4977376 0.5972851 0.6507177
## L4  0.4247312 0.5116883 0.5353535 0.5753086 0.5069767 0.5761905 0.5699208
## L5  0.4733179 0.5273973 0.5701754 0.5860566 0.4678112 0.5560345 0.5687646
## L6  0.5289673 0.5091384 0.5902439 0.6009732 0.5553047 0.6009390 0.6189258
## L7  0.5365854 0.5601093 0.5760000 0.5840000 0.5356265 0.5989848 0.6145251
## L8  0.5828571 0.6160458 0.6312849 0.6242938 0.5954198 0.6631579 0.6666667
## L9  0.7034700 0.7324841 0.7343750 0.7197452 0.7412399 0.7410714 0.7301038
##           L22       L23       L24        L3        L4        L5        L6
## A10                                                                      
## A11                                                                      
## A12                                                                      
## A13                                                                      
## A16                                                                      
## A2                                                                       
## A26                                                                      
## A27                                                                      
## A28                                                                      
## A3                                                                       
## A30                                                                      
## A31                                                                      
## A32                                                                      
## A33                                                                      
## A34                                                                      
## A4                                                                       
## A5                                                                       
## A6                                                                       
## A7                                                                       
## A8                                                                       
## A9                                                                       
## C1                                                                       
## C10                                                                      
## C11                                                                      
## C12                                                                      
## C2                                                                       
## C22                                                                      
## C23                                                                      
## C24                                                                      
## C25                                                                      
## C26                                                                      
## C27                                                                      
## C28                                                                      
## C29                                                                      
## C3                                                                       
## C30                                                                      
## C31                                                                      
## C32                                                                      
## C34                                                                      
## C35                                                                      
## C36                                                                      
## C37                                                                      
## C38                                                                      
## C39                                                                      
## C4                                                                       
## C40                                                                      
## C5                                                                       
## C6                                                                       
## C7                                                                       
## C8                                                                       
## C9                                                                       
## L1                                                                       
## L10                                                                      
## L11                                                                      
## L12                                                                      
## L14                                                                      
## L15                                                                      
## L18                                                                      
## L19                                                                      
## L2                                                                       
## L20                                                                      
## L21                                                                      
## L22                                                                      
## L23 0.5380952                                                            
## L24 0.5807963 0.5201238                                                  
## L3  0.5904366 0.6146096 0.6584158                                        
## L4  0.5892473 0.5844504 0.5983827 0.5273632                              
## L5  0.5753425 0.5849057 0.6200466 0.5374449 0.4455206                    
## L6  0.6202532 0.6047745 0.6332454 0.5714286 0.5091864 0.5150812          
## L7  0.6322870 0.5865103 0.6260870 0.5706806 0.4633431 0.5210918 0.5659341
## L8  0.6744731 0.6355140 0.6645963 0.5819209 0.5321101 0.5954198 0.6224784
## L9  0.7760814 0.6828358 0.7030075 0.7514970 0.7049180 0.7479893 0.7072368
##            L7        L8
## A10                    
## A11                    
## A12                    
## A13                    
## A16                    
## A2                     
## A26                    
## A27                    
## A28                    
## A3                     
## A30                    
## A31                    
## A32                    
## A33                    
## A34                    
## A4                     
## A5                     
## A6                     
## A7                     
## A8                     
## A9                     
## C1                     
## C10                    
## C11                    
## C12                    
## C2                     
## C22                    
## C23                    
## C24                    
## C25                    
## C26                    
## C27                    
## C28                    
## C29                    
## C3                     
## C30                    
## C31                    
## C32                    
## C34                    
## C35                    
## C36                    
## C37                    
## C38                    
## C39                    
## C4                     
## C40                    
## C5                     
## C6                     
## C7                     
## C8                     
## C9                     
## L1                     
## L10                    
## L11                    
## L12                    
## L14                    
## L15                    
## L18                    
## L19                    
## L2                     
## L20                    
## L21                    
## L22                    
## L23                    
## L24                    
## L3                     
## L4                     
## L5                     
## L6                     
## L7                     
## L8  0.5387205          
## L9  0.6367188 0.6444444

Using qualitative distances

Now you can run the ordination function in Phyloseq and then plot it.

qual_ord <- ordinate(physeq.rarefied, method="PCoA", distance=jac_dist)
plot_jac <- plot_ordination(physeq.rarefied, qual_ord, color="Name", title='Jaccard') + theme(aspect.ratio=1) + geom_point(size=4)
plot_jac

Using quantitative distances

For quantitative distance use the Bray-Curtis method:

bc_dist <- distance(physeq.rarefied, method = "bray", binary = FALSE)

Now run ordination on the quantitative distances and plot them

quant_ord <- ordinate(physeq.rarefied, method="PCoA", distance=bc_dist)
plot_bc <- plot_ordination(physeq.rarefied, quant_ord, color="Name", title='Bray-Curtis') + theme(aspect.ratio=1) + geom_point(size=3)
plot_bc

plot_Weevils_parasitization <- plot_ordination(physeq.rarefied, quant_ord, color = "Name", shape = 'Parasitization', title = "Jaccard Weevils and Parasitization") + theme(aspect.ratio = 1) + geom_point(size=1)
plot_Weevils_parasitization

plot_Weevils_location <- plot_ordination(physeq.rarefied, quant_ord, color = "Name", shape = 'Site', title = "Jaccard Weevils and Sites") + theme(aspect.ratio = 1) + geom_point(size=1)
plot_Weevils_location

# Permutational ANOVA

adonis(bc_dist ~ sample_data(physeq.rarefied)$Name)
## 
## Call:
## adonis(formula = bc_dist ~ sample_data(physeq.rarefied)$Name) 
## 
## Permutation: free
## Number of permutations: 999
## 
## Terms added sequentially (first to last)
## 
##                                   Df SumsOfSqs MeanSqs F.Model      R2 Pr(>F)
## sample_data(physeq.rarefied)$Name  2    10.647  5.3235  26.124 0.42739  0.001
## Residuals                         70    14.264  0.2038         0.57261       
## Total                             72    24.912                 1.00000       
##                                      
## sample_data(physeq.rarefied)$Name ***
## Residuals                            
## Total                                
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
adonis(bc_dist ~ sample_data(physeq.rarefied)$Parasitization)
## 
## Call:
## adonis(formula = bc_dist ~ sample_data(physeq.rarefied)$Parasitization) 
## 
## Permutation: free
## Number of permutations: 999
## 
## Terms added sequentially (first to last)
## 
##                                             Df SumsOfSqs MeanSqs F.Model
## sample_data(physeq.rarefied)$Parasitization  1    0.4248 0.42480  1.2317
## Residuals                                   71   24.4868 0.34488        
## Total                                       72   24.9116                
##                                                  R2 Pr(>F)
## sample_data(physeq.rarefied)$Parasitization 0.01705  0.275
## Residuals                                   0.98295       
## Total                                       1.00000
adonis(bc_dist ~ sample_data(physeq.rarefied)$Site)
## 
## Call:
## adonis(formula = bc_dist ~ sample_data(physeq.rarefied)$Site) 
## 
## Permutation: free
## Number of permutations: 999
## 
## Terms added sequentially (first to last)
## 
##                                   Df SumsOfSqs MeanSqs F.Model      R2 Pr(>F)
## sample_data(physeq.rarefied)$Site  2    4.4036 2.20178  7.5153 0.17677  0.001
## Residuals                         70   20.5080 0.29297         0.82323       
## Total                             72   24.9116                 1.00000       
##                                      
## sample_data(physeq.rarefied)$Site ***
## Residuals                            
## Total                                
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
adonis(bc_dist ~ sample_data(physeq.rarefied)$Sex)
## 
## Call:
## adonis(formula = bc_dist ~ sample_data(physeq.rarefied)$Sex) 
## 
## Permutation: free
## Number of permutations: 999
## 
## Terms added sequentially (first to last)
## 
##                                  Df SumsOfSqs MeanSqs F.Model      R2 Pr(>F)
## sample_data(physeq.rarefied)$Sex  2    0.6609 0.33044 0.95383 0.02653  0.507
## Residuals                        70   24.2507 0.34644         0.97347       
## Total                            72   24.9116                 1.00000

Subsetting by taxonomy

Rickettisia_subset

Ricket <- subset_taxa(physeq.rarefied, Order=='Rickettsiales')
Ricket
## phyloseq-class experiment-level object
## otu_table()   OTU Table:         [ 4 taxa and 73 samples ]
## sample_data() Sample Data:       [ 73 samples by 4 sample variables ]
## tax_table()   Taxonomy Table:    [ 4 taxa by 7 taxonomic ranks ]
plot_bar(Ricket, fill = 'Genus')

plot_bar(Ricket, fill = 'Family')

barplot5 <- plot_bar(Ricket, x="Parasitization", fill = 'Genus') + facet_wrap(~Name, scales = "free_x", nrow = 1)
barplot5

Subsampling for Buchnera genus

Enterobacte <- subset_taxa(physeq.rarefied, Family=='Enterobacteriaceae')
Enterobacte
## phyloseq-class experiment-level object
## otu_table()   OTU Table:         [ 23 taxa and 73 samples ]
## sample_data() Sample Data:       [ 73 samples by 4 sample variables ]
## tax_table()   Taxonomy Table:    [ 23 taxa by 7 taxonomic ranks ]
plot_bar(Enterobacte, fill = 'Genus')

barplot6 <- plot_bar(Enterobacte, x="Parasitization", fill = 'Genus') + facet_wrap(~Name, scales = "free_x", nrow = 1)
barplot6

alphab <- subset_taxa(physeq.rarefied, Class=='Alphaproteobacteria')
plot_bar(alphab, fill = 'Family')

Subsampling for CRW

CRW <- subset_samples(physeq, Name=='Crw')
CRW
## phyloseq-class experiment-level object
## otu_table()   OTU Table:         [ 1142 taxa and 30 samples ]
## sample_data() Sample Data:       [ 30 samples by 4 sample variables ]
## tax_table()   Taxonomy Table:    [ 1142 taxa by 7 taxonomic ranks ]
Crw <-plot_bar(CRW, fill = 'Phylum')
Crw

crw.rarefied <- rarefy_even_depth(CRW, rngseed=1, sample.size = 0.9*min(sample_sums(CRW)), replace = F)
## `set.seed(1)` was used to initialize repeatable random subsampling.
## Please record this for your records so others can reproduce.
## Try `set.seed(1); .Random.seed` for the full vector
## ...
## 410OTUs were removed because they are no longer 
## present in any sample after random subsampling
## ...

Now, calculate the distance and ordination

crw_dist <- distance(crw.rarefied, method = 'bray', binary=FALSE)

crw_ord <- ordinate(crw.rarefied, method="PCoA", distance=crw_dist)

plot the ordination

plot_crw <- plot_ordination(crw.rarefied, crw_ord, color='Site',shape='Parasitization', title="CRW Parasitization and Site PCoA")+
theme(aspect.ratio=1)+ geom_point(size=4)

plot_crw

Anova CRW parasitization

adonis(crw_dist ~ sample_data(crw.rarefied)$Parasitization)
## 
## Call:
## adonis(formula = crw_dist ~ sample_data(crw.rarefied)$Parasitization) 
## 
## Permutation: free
## Number of permutations: 999
## 
## Terms added sequentially (first to last)
## 
##                                          Df SumsOfSqs MeanSqs F.Model     R2
## sample_data(crw.rarefied)$Parasitization  1    0.1253 0.12528 0.62392 0.0218
## Residuals                                28    5.6225 0.20080         0.9782
## Total                                    29    5.7477                 1.0000
##                                          Pr(>F)
## sample_data(crw.rarefied)$Parasitization  0.626
## Residuals                                      
## Total

Not significant

adonis(crw_dist ~ sample_data(crw.rarefied)$Site)
## 
## Call:
## adonis(formula = crw_dist ~ sample_data(crw.rarefied)$Site) 
## 
## Permutation: free
## Number of permutations: 999
## 
## Terms added sequentially (first to last)
## 
##                                Df SumsOfSqs MeanSqs F.Model      R2 Pr(>F)  
## sample_data(crw.rarefied)$Site  1    0.8377 0.83771  4.7772 0.14575  0.011 *
## Residuals                      28    4.9100 0.17536         0.85425         
## Total                          29    5.7477                 1.00000         
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Significant

adonis(crw_dist ~ sample_data(crw.rarefied)$Sex)
## 
## Call:
## adonis(formula = crw_dist ~ sample_data(crw.rarefied)$Sex) 
## 
## Permutation: free
## Number of permutations: 999
## 
## Terms added sequentially (first to last)
## 
##                               Df SumsOfSqs MeanSqs F.Model      R2 Pr(>F)
## sample_data(crw.rarefied)$Sex  1    0.2057 0.20566  1.0391 0.03578  0.321
## Residuals                     28    5.5421 0.19793         0.96422       
## Total                         29    5.7477                 1.00000

Not significant

Subsampling LW

LW <- subset_samples(physeq, Name=='Lw')
LW
## phyloseq-class experiment-level object
## otu_table()   OTU Table:         [ 1142 taxa and 21 samples ]
## sample_data() Sample Data:       [ 21 samples by 4 sample variables ]
## tax_table()   Taxonomy Table:    [ 1142 taxa by 7 taxonomic ranks ]
plot_bar(LW, fill = 'Phylum')

lw.rarefied <- rarefy_even_depth(LW, rngseed = 1, sample.size = 0.9*min(sample_sums(LW)), replace = F)
## `set.seed(1)` was used to initialize repeatable random subsampling.
## Please record this for your records so others can reproduce.
## Try `set.seed(1); .Random.seed` for the full vector
## ...
## 333OTUs were removed because they are no longer 
## present in any sample after random subsampling
## ...
lw_dist <- distance(lw.rarefied, method = 'bray', binary=FALSE)

lw_ord <- ordinate(lw.rarefied, method="PCoA", distance=lw_dist)
plot_lw <- plot_ordination(lw.rarefied, lw_ord, color='Site',shape='Parasitization', title="LW Parasitization and Site PCoA")+
theme(aspect.ratio=1)+ geom_point(size=4)

plot_lw

Anova LW

adonis(lw_dist ~ sample_data(lw.rarefied)$Parasitization)
## 
## Call:
## adonis(formula = lw_dist ~ sample_data(lw.rarefied)$Parasitization) 
## 
## Permutation: free
## Number of permutations: 999
## 
## Terms added sequentially (first to last)
## 
##                                         Df SumsOfSqs MeanSqs F.Model      R2
## sample_data(lw.rarefied)$Parasitization  1    0.4119 0.41189  2.1194 0.10035
## Residuals                               19    3.6926 0.19435         0.89965
## Total                                   20    4.1045                 1.00000
##                                         Pr(>F)
## sample_data(lw.rarefied)$Parasitization  0.115
## Residuals                                     
## Total

Not significant

adonis(lw_dist ~ sample_data(lw.rarefied)$Site)
## 
## Call:
## adonis(formula = lw_dist ~ sample_data(lw.rarefied)$Site) 
## 
## Permutation: free
## Number of permutations: 999
## 
## Terms added sequentially (first to last)
## 
##                               Df SumsOfSqs MeanSqs F.Model      R2 Pr(>F)   
## sample_data(lw.rarefied)$Site  1    1.1101  1.1101  7.0437 0.27046  0.002 **
## Residuals                     19    2.9944  0.1576         0.72954          
## Total                         20    4.1045                 1.00000          
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Significant, check how many samples you have per site, too low sample size will skew the result

adonis(lw_dist ~ sample_data(lw.rarefied)$Sex)
## 
## Call:
## adonis(formula = lw_dist ~ sample_data(lw.rarefied)$Sex) 
## 
## Permutation: free
## Number of permutations: 999
## 
## Terms added sequentially (first to last)
## 
##                              Df SumsOfSqs MeanSqs F.Model      R2 Pr(>F)
## sample_data(lw.rarefied)$Sex  1    0.2030 0.20298 0.98852 0.04945  0.383
## Residuals                    19    3.9015 0.20534         0.95055       
## Total                        20    4.1045                 1.00000

Not significant

Subsampling for ASW

ASW <- subset_samples(physeq, Name=='Asw')
ASW
## phyloseq-class experiment-level object
## otu_table()   OTU Table:         [ 1142 taxa and 22 samples ]
## sample_data() Sample Data:       [ 22 samples by 4 sample variables ]
## tax_table()   Taxonomy Table:    [ 1142 taxa by 7 taxonomic ranks ]
plot_bar(ASW, fill = 'Phylum')

Asw.rarefied <- rarefy_even_depth(ASW, rngseed = 1, sample.size = 0.9*min(sample_sums(ASW)), replace = F)
## `set.seed(1)` was used to initialize repeatable random subsampling.
## Please record this for your records so others can reproduce.
## Try `set.seed(1); .Random.seed` for the full vector
## ...
## 109OTUs were removed because they are no longer 
## present in any sample after random subsampling
## ...
plot_bar(Asw.rarefied, fill = 'Phylum')

asw_dist <- distance(Asw.rarefied, method = 'bray', binary=FALSE)

asw_ord <- ordinate(Asw.rarefied, method="PCoA", distance=asw_dist)
plot_asw <- plot_ordination(Asw.rarefied, asw_ord, color='Parasitization',shape='Sex', title="ASW Parasitization and Sex PCoA")+
theme(aspect.ratio=1)+ geom_point(size=4)

plot_asw

adonis(asw_dist ~ sample_data(Asw.rarefied)$Parasitization)
## 
## Call:
## adonis(formula = asw_dist ~ sample_data(Asw.rarefied)$Parasitization) 
## 
## Permutation: free
## Number of permutations: 999
## 
## Terms added sequentially (first to last)
## 
##                                          Df SumsOfSqs MeanSqs F.Model      R2
## sample_data(Asw.rarefied)$Parasitization  1    0.8993 0.89926   5.117 0.20373
## Residuals                                20    3.5148 0.17574         0.79627
## Total                                    21    4.4140                 1.00000
##                                          Pr(>F)  
## sample_data(Asw.rarefied)$Parasitization  0.011 *
## Residuals                                        
## Total                                            
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Significant Check sample size

adonis(asw_dist ~ sample_data(Asw.rarefied)$Sex)
## 
## Call:
## adonis(formula = asw_dist ~ sample_data(Asw.rarefied)$Sex) 
## 
## Permutation: free
## Number of permutations: 999
## 
## Terms added sequentially (first to last)
## 
##                               Df SumsOfSqs MeanSqs F.Model     R2 Pr(>F)
## sample_data(Asw.rarefied)$Sex  2    0.4816 0.24078  1.1634 0.1091  0.317
## Residuals                     19    3.9325 0.20697         0.8909       
## Total                         21    4.4140                 1.0000